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Biomedical subjects

J Yee

Publications and source records attributed to J Yee.

At least 19 recordsLinked to original sources
PubMed1995

Transient transfection and expression of firefly luciferase in Giardia lamblia.

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.

Animals↗
PubMed1995

Passive immunization of rhesus macaques against SIV infection and disease.

To evaluate the role of humoral immunity against simian immunodeficiency virus (SIV), we tested whether passive immunization with plasma from SIVmac251 vaccine-protected or healthy infected animals would protect rhesus monkeys against intravenous infection with ten 50% animal infectious doses of the cell-free homologous virus. The challenge dose of this SIVmac251 virus stock had previously caused persistent infection in all (21 of 21) nonimmunized controls. A plasma pool was obtained from a donor that had been immunized with an inactivated whole SIVmac251 vaccine produced in human T cells. This plasma pool contained low levels of SIVmac binding and neutralizing antibody but had a high titer of antibodies recognizing human cell proteins. Given 4 or 18 hr before intravenous challenge, this plasma completely protected three of eight recipients from infection and delayed virus detection in one recipient. The five unprotected animals had only a transient or undetectable p27 antigenemia and low virus load in their PBMCs, and all survived at least 7 months after infection. By contrast, no protection was observed in 6 monkeys given inactivated, pooled plasma or purified immunoglobulin (Ig) from healthy SIVmac251-infected animals. This plasma pool and the Ig preparation contained high levels of SIV-binding and neutralizing antibody but no reactivity to human cellular components. Five of the six recipients had persistent antigenemia after challenge and four died acutely from simian AIDS in 4-7 months. These studies suggest that passive transfer of antibody to human cellular antigens can confer protection against SIVmac whereas passive transfer of neutralizing antibodies without human cellular antibodies does not protect against the homologous virus and may enhance infection.

Animals↗
PubMed1995

The delayed hypersensitivity response and host resistance in surgical patients. 20 years later.

OBJECTIVE: A 20-year follow-up was conducted on research into the implications of a lack of a delayed-type hypersensitivity (DTH) skin test response among surgical patients. SUMMARY BACKGROUND DATA: The authors' original report showed that a failed DTH response was associated with increased hospital mortality, but the role of specific and nonspecific host defense elements, comorbid factors, nutritional supplementation, and the mechanism for anergy in this adverse outcome was unknown. METHODS: A data base of 4292 patients was analyzed and reported on individual studies designed to answer some of the above questions. RESULTS: Prospective studies showed a strong association between the DTH response and mortality: reactive patients, 2.9% (75/2576); anergic patients, 20.9% (239/1142, chi square = 265, p < 0.0000001). Antibody response to protein antigens was reduced in anergic patients. Antibody response to polysaccharide antigens was normal in all patients. The hallmark of anergy is a lack of T cells in the skin, as measured by mRNA signal (CD3) for T cells. The nonspecific component of host defense, as measured by circulating and exudate polymorphonuclear cell function, showed no statistically significant difference between elective reactive and elective anergic patients. Notwithstanding some mild malnutrition in anergic patients, parental nutrition failed to correct the DTH response or many of the cellular immune functions measured. CONCLUSIONS: Over the last 5 years, because of a reduction in overall patient mortality, the contribution of a reduced DTH response to septic related mortality has lost statistical significance in elective surgical patients. A reduced DTH response maintains its strong association to sepsis-related mortality in intensive care/trauma patients, and this is the group on which future research efforts should be concentrated.

Aged↗
PubMed1995

Developmentally regulated expression of a Giardia lamblia cyst wall protein gene.

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100-fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation-specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine-rich polypeptide of M(r) 26,000 that contains 5.3 tandemly arranged copies of a degenerate 24-amino-acid repeat. A hydrophobic amino-terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

Amino Acid Sequence↗
PubMed1995

Gastrointestinal manifestations of AIDS.

As the AIDS epidemic continues to grow, it becomes increasingly important that physicians are aware of the gastrointestinal and abdominal diseases specific to this group of patients. Most AIDS patients exhibit gastrointestinal symptoms at some time during the course of their disease. Clinical AIDS is often determined by identifying an opportunistic infection or neoplasm of the gastrointestinal tract. Radiology often plays a key role in helping to determine the diagnosis as well as in directing the management.

AIDS-Related Opportunistic Infections↗
PubMed1994

CYP1A1 genetic polymorphisms and in situ colorectal cancer.

Numerous studies have associated colorectal adenoma with smoking and large bowel cancer with consumption of foods potentially containing polycyclic aromatic hydrocarbons. Enhanced metabolic activation of polycyclic aromatic hydrocarbons has recently been observed in homozygotes for a MspI mutation in the 3'-end of CYP1A1. We conducted a population-based case-control study to investigate whether CYP1A1 polymorphisms were related to colorectal cancer risk. Using polymerase chain reaction-based methods, we assessed the frequency of the MspI polymorphism in the 3'-end of CYP1A1 and another mutation in exon 7 of the gene (Ile-Val polymorphism) among 43 patients with in situ adenocarcinoma of the large bowel and 129 population controls. Homozygosity for the MspI mutant genotype was found to be positively associated with in situ colorectal cancer in Japanese (P = 0.008) and Hawaiians/part-Hawaiians (P < 0.001), whereas the study lacked power to detect a similar association in Caucasians. The odds ratio for the homozygous variant genotype compared to the heterozygous and wild-type genotypes was 7.9 (95% confidence interval, 1.4-44.4) in Japanese. A similar association was suggested for the exon 7 mutation homozygosity in Japanese, as the two polymorphisms are in genetic disequilibrium. Thus, this study suggests a potentially important role for CYP1A1 and polycyclic aromatic hydrocarbons in the etiology of colorectal cancer in populations with a high gene frequency.

Adult↗
PubMed1994

The local role of tumor necrosis factor alpha in the modulation of neutrophil function at sites of inflammation.

OBJECTIVE: To examine the hypothesis that tumor necrosis factor alpha (TNF-alpha) is an important local modulator of neutrophil function in the inflammatory microenvironment. DESIGN: In vitro studies of host defense. PATIENTS: A volunteer sample of healthy subjects. INTERVENTION: Exudative neutrophils were collected from skin-blister chambers and functionally compared with blood neutrophils. METHODS: Tumor necrosis factor alpha levels at sites of inflammation and neutrophil exudation were determined and compared with serum concentrations. Flow cytometry was used to evaluate neutrophil microbicidal activity and N-formyl-methionyl-leucyl-phenylalanine-induced changes in intracellular calcium and superoxide production. In vitro TNF-alpha was used to evaluate the nature and dose response of TNF-alpha-induced changes in neutrophil function. RESULTS: Exudative neutrophils have an increased responsiveness to subsequent N-formyl-methionyl-leucyl-phenylalanine stimulation, as determined by changes in intracellular calcium. Microbicidal activity and superoxide production are also up-regulated compared with circulating neutrophils. The exudative microenvironment contains TNF-alpha at local levels that are capable of significantly enhancing neutrophil host defense. CONCLUSIONS: Tumor necrosis factor alpha may serve to enhance neutrophil function at sites of inflammation. Neutrophils become more cytotoxic and have an enhanced ability to respond to weak environmental signals.

Blood Bactericidal Activity↗
PubMed1994

A yeast artificial chromosome clone map of the Drosophila genome.

We describe the mapping of 979 randomly selected large yeast artificial chromosome (YAC) clones of Drosophila DNA by in situ hybridization to polytene chromosomes. Eight hundred and fifty-five of the clones are euchromatic and have primary hybridization sites in the banded portions of the polytene chromosomes, whereas 124 are heterochromatic and label the chromocenter. The average euchromatic clone contains about 211 kb and, at its primary site, labels eight or nine contiguous polytene bands. Thus, the extent as well as chromosomal position of each clone has been determined. By direct band counts, we estimate our clones provide about 76% coverage of the euchromatin of the major autosomes, and 63% coverage of the X. When previously reported YAC mapping data are combined with ours, euchromatic coverage is extended to about 90% for the autosomes and 82% for the X. The distribution of gap sizes in our map and the coverage achieved are in good agreement with expectations based on the assumption of random coverage, indicating that euchromatic clones are essentially randomly distributed. However, certain gaps in coverage, including the entire fourth chromosome euchromatin, may be significant. Heterochromatic sequences are underrepresented among the YAC clones by two to three fold. This may result, at least in part, from underrepresentation of heterochromatic sequences in adult DNA (the source of most of the clones analyzed), or from clone instability.

Animals↗
PubMed1994

Infectious esophagitis.

Infectious esophagitis is most often seen in patients with impaired host resistance. It has become a particular problem in the growing AIDS population. The three most commonly encountered opportunistic infections of the esophagus are Candida albicans, herpes simplex virus, and cytomegalovirus. Candida is the single leading cause of infectious esophagitis. Tuberculous and bacterial esophagitis and other unusual fungal infections of the esophagus are uncommon.

Candidiasis↗
PubMed1993

Neutrophil priming by lipopolysaccharide involves heterogeneity in calcium-mediated signal transduction. Studies using fluo-3 and flow cytometry.

Bacterial LPS is known to prime neutrophils for enhanced responses to subsequent stimulation by agonists such as FMLP. The purpose of this study was to determine whether priming is due to a uniform enhancement of function in all cells or to a recruitment of previously unresponsive neutrophils. Results from initial experiments confirmed the findings of other investigators and served to validate our methodology. We demonstrated that LPS-primed neutrophils had: 1) augmented superoxide anion production after FMLP or PMA stimulation; 2) increased FMLP-induced f-actin formation; 3) enhanced surface expression of CD11b, CD14, and the FMLP receptor; 4) caused higher baseline concentrations of intracellular calcium ([Ca2+]i); 5) caused greater elevations of [Ca2+]i after FMLP stimulation; and that 6) the priming effect of LPS on superoxide anion production and f-actin polymerization could be blocked by the [Ca2+]i chelator bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. In subsequent experiments, we determined that at low concentrations of FMLP, LPS augmented the overall responsiveness of a population of neutrophils by causing a subpopulation of cells to become highly responsive and able to generate changes in [Ca2+]i upon low level stimulation. Heterogeneity in calcium-mediated signal transduction among neutrophils may be important in the fine control of the nonspecific immune system in response to weak environmental signals.

Actins↗
PubMed1992

Isolation and characterization of a NADP-dependent glutamate dehydrogenase gene from the primitive eucaryote Giardia lamblia.

Giardia lamblia is believed to be the earliest branching derivative from the eucaryotic lineage. Genomic and cDNA clones encoding the giardia NADP-dependent glutamate dehydrogenase have been isolated and characterized. Southern hydridization using genomic DNA indicates that the gene encoding this activity is unique and single copy. Primer extension, S1 nuclease protection, and genomic and cDNA sequence analysis demonstrate that gene transcripts are initiated within a conserved AT-rich sequence element immediately preceding the ATG translation initiation codon and the short 5' untranslated region is not extended by transsplicing. The open reading frame is 1350 nucleotides in length and encodes a protein of 449 amino acids. The reading frame is not interrupted by introns and the primary transcript is probably not subjected to RNA editing. In the strictly anaerobic metabolism of giardia, NADP-dependent glutamate dehydrogenase activity participates along with alanine aminotransferase, in the cyclic dissipation of reducing equivalents (NADPH) through the conversion of pyruvate to alanine. The deduced amino acid sequence of the giardia protein exhibits substantial homology to numerous fungal and eubacterial NADP-dependent glutamate dehydrogenases. Comparisons of alignment gap positions and amino acid identities indicate that the giardia sequence is at least as similar or more similar to the eubacterial sequence than it is to the fungal sequence. This supports the hypothesis that giardia diverged very early from the eucaryotic lineage.

Amino Acid Sequence↗
PubMed1992

Development of a monoclonal antibody-based p24 capsid antigen detection assay for HTLV-I, HTLV-II, and STLV-I infection.

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and employed to detect p24 capsid antigen from human T-cell lymphotropic viruses type I and II (HTLV-I, HTLV-II), simian T-cell lymphotropic virus type I (STLV-I)-infected cell lines, and from mononuclear cell cocultures of HTLV-infected humans and STLV-I infected monkeys. A monoclonal antibody specific for HTLV p24 and p53 capsid antigens was coated onto 96-well microtiter plates to capture HTLV/STLV antigen. Captured antigen was then detected by the addition of a polyclonal, biotinylated human anti-HTLV-I antibody, and color developed with tetramethyl benzidine/H2O2 substrate. As little as 15 pg/ml of HTLV-I p24 antigen could be detected in this assay. Culture supernatants from HTLV-I-infected cell lines (HUT-102, MT-2, C5/MJ, HTLV-II-infected cell lines (Mo-T, Mo-B, PanG 12.1, NRA) and STLV-I-infected cell lines (Matsu, NEPC M39) were all positive in the assay. In addition, p24 was detected from peripheral blood mononuclear cell (PBMC) cocultures of 8 of 8 (100%) HTLV-I diseased patients, 14 of 20 (70%) HTLV-I and HTLV-II-infected, asymptomatic persons, and 8 of 8 (100%) STLV-I-infected, asymptomatic monkeys. Culture supernatants of cells infected with human immunodeficiency virus type (HIV-1), simian immunodeficiency virus (SIV), Chlamydia trachomatis, cytomegalovirus (CMV), herpes simplex I and II (HSV), feline leukemia virus (FELV), bovine leukemia virus (BLV), and bovine immunodeficiency virus (BIV) were all negative. Similarly, normal human peripheral blood mononuclear cells and uninfected, transformed human T cells, were also negative in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Serologic confirmation of simian T-lymphotropic virus type I infection by using immunoassays developed for human T-lymphotropic virus antibody detection.

Serum specimens from diverse species of Old World monkeys, categorized as seropositive (n = 97) or seronegative (n = 23) for human T-lymphotropic virus (HTLV) infection, were tested by using recombinant env-spiked Western immunoblot assays and synthetic peptide assays for simultaneous detection and discrimination of simian T-lymphotropic virus (STLV) infection. Of the 97 seropositive specimens, 93 reacted with the recombinant transmembrane (r21env) protein and 90 reacted with a recombinant, MTA-1, derived from the central region of the external glycoprotein of HTLV-I (rgp46env), thus yielding test sensitivities of 96 and 93%, respectively. While 1 of the 23 negative monkey specimens reacted with r21env, none reacted with rgp46env, for overall specificities of 96 and 100%, respectively. Analysis of synthetic peptide-based immunoassays demonstrated that while 85 of 97 (88%) seropositive specimens reacted with HTLV-I-specific epitope (p19gag), none of the specimens reacted with HTLV-II-specific epitope (gp52env). These results show that recombinant envelope-spiked Western blots provide a simple means for serologic confirmation of STLV-I infection and that type-specific synthetic peptides can be used to confirm the virus type in seropositive monkey specimens.

Animals↗
PubMed1991

Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization.

Defensins (molecular weight 3500 to 4000) act in the mammalian immune response by permeabilizing the plasma membranes of a broad spectrum of target organisms, including bacteria, fungi, and enveloped viruses. The high-resolution crystal structure of defensin HNP-3 (1.9 angstrom resolution, R factor 0.19) reveals a dimeric beta sheet that has an architecture very different from other lytic peptides. The dimeric assembly suggests mechanisms by which defensins might bind to and permeabilize the lipid bilayer.

Amino Acid Sequence↗
PubMed1991

Indomethacin inhibition of middle ear bone resorption.

Localized osteoclastic bone resorption is responsible for the pathological changes within the middle and inner ear, which result in hearing loss and vertigo in chronic otitis media and otosclerosis. The local control of osteoclastic bone resorption is incompletely understood. Various small, locally active molecules, cytokines, have been shown to affect resorptive processes. Additionally, prostaglandins and their inhibitors have been shown to modulate the resorptive process in a number of in vitro studies. In this study, indomethacin, a cyclooxygenase inhibitor, was tested in a model of localized bone resorption, the pressurized gerbil bulla. After the experimental period, indomethacin was found to inhibit the number of osteoclasts and the resorptive area on the inner surface of the bulla. Therefore, it is likely that endogenous cyclooxygenase metabolites are intermediates in the sequence of cellular events, which results in localized bone resorption as in some systemic models.

Animals↗
PubMed1991

Clostridium difficile disease in a department of surgery. The significance of prophylactic antibiotics.

A clustering of Clostridium difficile-associated disease in a department of surgery prompted a program of infection control and the evaluation of contributing factors. Fifty patients had diarrhea and positive assays for C difficile cytotoxin during the study period. Twenty-one of the 36 cases that developed among patients admitted to the surgical services occurred on two adjacent general surgery wards that shared attending surgeons and house staff. Perioperative prophylactic antibiotics predated C difficile-associated disease in 20 patients, 12 of whom had short courses (less than 24 hours). Symptoms were typically nonspecific and early diagnosis may be difficult. Incidence remained high, despite infection control measures, until the coincidental closure of two surgical wards. Clostridium difficile-associated disease is a nosocomial infection that can be associated with short courses of prophylactic antibiotics. Recommendations regarding the use of perioperative prophylaxis should recognize C difficile-associated disease as a significant potential complication.

Adult↗