Biomedical subjects

D M Phillips

Publications and source records attributed to D M Phillips.

At least 73 records · Page 4Linked to original sources

PubMed1993

PECAM-1 is required for transendothelial migration of leukocytes.

Platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) is crucial to the process of leukocyte transmigration through intercellular junctions of vascular endothelial cells. A monoclonal antibody to PECAM, or recombinant soluble PECAM, blocks transendothelial migration of monocytes by 70-90%. Pretreating either the monocytes or the endothelial junctions with antibody blocks transmigration. If the endothelium is first activated by cytokines, anti-PECAM antibody or soluble recombinant PECAM again block transmigration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type. Light and electron microscopy reveal that leukocytes blocked in transmigration remain tightly bound to the apical surface of the endothelial cell, precisely over the intercellular junction. Thus, the process of leukocyte emigration can be dissected into three successive stages: rolling, mediated by the selectin class of adhesion molecules; tight adhesion, mediated by the leukocyte integrins and their endothelial cell counter-receptors; and now transmigration, which, based on these studies, requires PECAM-1.

Antibodies, Monoclonal↗

PubMed1993

Hybrid sterility-6: a mouse t complex locus controlling sperm flagellar assembly and movement.

Mouse t haplotypes alter sperm differentiation, resulting in abnormal sperm movement and sterility. In previous studies, a locus responsible for hybrid sterility in the genus Mus,Hst-4, was mapped to the distal inversion of the t complex on chromosome 17. Here we report the identification and characterization of two additional hybrid sterility loci, Hst-5 and Hst-6, that map to the same inversion. We further show that an abnormality in sperm flagellar curvature deriving from interactions between t haplotypes and the M. spretus allele of Hst-6 is indistinguishable from one exhibited by sperm from mice carrying two t haplotypes. Additionally, we demonstrate that this latter phenotype maps to the distal inversion of t haplotypes. Morphological and functional studies of Hst-6 mutant sperm also imply that the product(s) of Hst-6 is a spermatogenic-specific protein, important for assembly and function of the sperm axoneme. Thus, Hst-6 provides direct access to the molecular basis of t haplotype-specific alterations in sperm function that emanate from the t complex distal inversion.

Alleles↗

PubMed1993

Fertilization and early development of rat oocytes induced to mature by forskolin.

Forskolin has been shown to successfully induce maturation of rat oocytes as assessed by morphological markers. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by forskolin (10(-4) M, group A) in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. Oocytes exposed in vitro to either luteinizing hormone (LH, 5 micrograms/ml, group B) or a GnRH agonist analogue (10(-7) M, group C) as well as oocytes that underwent maturation in vivo (group D), served as positive controls. We found that similar rates of fertilization were obtained in the experimental and all of the above mentioned control groups (A = 78.9 +/- 4.2%, B = 77.9 +/- 3.1%, C = 77.5 +/- 5.5% and D = 84.7 +/- 2.7%). Cleavage rate of fertilized eggs from group A was significantly higher than that of eggs from groups B & C, and similar to that of eggs from group D (A = 63.1 +/- 6.7%, B = 37.8 +/- 4.9%, C = 50.0 +/- 4.1%, D = 67.8 +/- 4.1%). Using functional parameters we hereby demonstrate that forskolin and LH are at least equally potent in producing fertilizable eggs that have a high potential of development into two cell embryos. These results further support the idea that cAMP is a mediator of LH action in inducing oocyte maturation.

Analysis of Variance↗

PubMed1993

Studies of adhesion of lymphocytic cells: implications for sexual transmission of human immunodeficiency virus.

Considerable evidence suggests that sexual transmission of human immunodeficiency virus (HIV) is mediated via mononuclear cells that can infect epithelia of the genital tract. We describe here an in vitro model that can be used to examine the mechanism of cell-to-cell transmission of this virus. We have employed the system to identify agents that may be effective in a vaginal formulation to prevent HIV transmission via sexual contact. We have previously shown that chronically HIV-infected mononucleocytes can infect CD4-negative epithelial monolayers in the following manner: adhesion, via multiple microvilli, of HIV-infected mononucleocyte-derived cells to epithelial monolayers activates rapid virion secretion. Virions are then shed from the attached surface of the infected lymphocyte into a partially enclosed, microvilli-laden space between the cells. The shedding results in uptake of the virus and epithelial cell infection as demonstrated by ultrastructural examination and in vitro virological techniques. In this report, we present evidence from time-lapse films that HIV-infected lymphocytes adhere to the epithelium for a few minutes and then shift position to another site on the epithelium. As a result, one infected lymphocyte appears to be able to sequentially infected several cells of the epithelial monolayer. Using a fluorescence-based cell-cell adhesion assay to examine the effect of seminal fluid and a variety of chemical compounds on lymphocyte-to-epithelial adherence, we found that seminal fluid significantly increases the number of lymphocytes adhering to epithelia. This suggests that semen can serve as an effective medium for cell-cell transmission of HIV. On the other hand, sulfated polysaccharides and glutathione effectively inhibit cell-cell adhesion. Since the cell-cell adhesion step is critical to epithelial cell infection by HIV, these results suggest that anti-cell adhesion compounds may be effective in a vaginal formulation to reduce the probability of HIV infection.

Cell Adhesion↗

PubMed1993

Factors that may regulate assembly of the mammalian sperm tail deduced from a mouse t complex mutation.

A unique pattern of aberrant sperm development occurs in laboratory mice (Mus domesticus) that have been made homozygous for an allele of an autosomal gene, Hst-6s, derived from another mouse species, Mus spretus. During the abnormal spermiogenic process of these sterile animals, the sperm tail fails to assemble normally, although a bud develops at the site of the centriole in early spermatids. The bud enlarges during sperm development and eventually contains elements of a mature sperm tail. Tubulin assembles primarily into single microtubules rather than doublets. These singlets form a circular array that is not oriented relative to the centriole. While dense fibers form in association with the singlet microtubules, the fibrous sheath components accumulate in a single irregular mass. These observations suggest that microtubule doublets are required for the organization of an axoneme. In addition, while dense fibers can form normally in association with singlet microtubules, the fibrous sheath requires the axoneme as a substrate for normal organization.

Animals↗

PubMed1993

Productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission.

The human cervix-derived epithelial cell line (ME180) used in this study displays a characteristics epithelial morphology, including numerous desmosomes, tonofilaments, and epidermal filaments. When T-cell lines infected with human immunodeficiency virus (HIV) are added to epithelial cultures, they rapidly adhere to the epithelial monolayer. Within a few minutes, the T cells shed numerous virions into narrow spaces formed between the epithelial cell and the adherent T cells. Virions subsequently enter the ME180 cells via large vesicles. A few days after infection, cytopathic effects and syncytium formation were observed. Infected clones of ME180 cells have remained infected for 8 months. p24 enzyme-linked immunosorbent assay and infectivity assays show that one subclone of the cell line produces virus titers equivalent to those of high-secreting HIV-infected T-cell lines. Electron microscopy reveals numerous virions budding from both the basal and apical surfaces of the epithelium. These observations suggest that cervical epithelium has the potential to serve as a site of HIV infection.

CD4 Antigens↗

PubMed1993

Differential regulation of steroidogenic enzymes during differentiation optimizes testosterone production by adult rat Leydig cells.

The postnatal differentiation of rat Leydig cells may be subdivided into three stages based on morphology and steroid production. The purpose of this study was to clarify the developmental mechanisms underlying increased testosterone production by measuring steady state levels of the mRNAs for three steroidogenic enzymes in isolated Leydig cells at each stage of differentiation. These include Leydig cell progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90. The steroidogenic enzymes were 1) cholesterol side-chain cleavage enzyme (CSCC), 2) 17 alpha-hydroxylase (P450-17 alpha), and 3) 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD). We report that levels of CSCC and P450-17 alpha mRNAs increase, whereas 3 alpha HSD mRNA levels decline during the course of Leydig cell differentiation. The levels of 3 alpha HSD mRNA were high in progenitor Leydig cells that appeared to contain little smooth endoplasmic reticulum and decreased in cells as smooth endoplasmic reticulum developed and other enzyme mRNAs increased. These observations suggest that the factors that regulate 3 alpha HSD mRNA levels are startlingly different from those that regulate the mRNA levels of CSCC and P450-17 alpha. We conclude that the progressive increase in the capacity of differentiating Leydig cells to produce testosterone can be explained in part by an increase in the activity of enzymes that synthesize testosterone (CSCC and P450-17 alpha) and a decrease in the activity of an enzyme that metabolizes testosterone and its precursors (3 alpha HSD).

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Practice and attitudes of primary care physicians in a rural southern state concerning HIV/AIDS patients.

In preparing for the development of a plan of care for persons with AIDS and other HIV related conditions, the HIV Services Planning Program, a joint effort of the Department of Family Medicine of the University of Mississippi Medical Center and the Bureau of Preventive Health of the Mississippi State Department of Health, conducted a survey of all primary care physicians throughout the state of Mississippi. The purpose of the survey was to determine health care services provided to persons with HIV/AIDS. Specific areas of interest included: (a) type of medical specialty; (b) location of primary practice; (c) services provided for persons with HIV/AIDS; and (d) utilization of universal blood and body fluid precautions, i.e., barrier techniques. Descriptive statistics were utilized in analyzing responses.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions.

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.

Androgens↗

PubMed1992

Mechanism of HIV spread from lymphocytes to epithelia.

Contact of human immunodeficiency virus (HIV)-infected MOLT-4 lymphocytes with epithelial cells derived from small intestine (I407; Intestine 407) resulted in a rapid polar budding of viral particles into an enclosed space formed by interdigitating microvilli of the contacting cells. Electron microscopy showed that released HIV was taken up into the mucosal cell via three independent mechanisms: (1) phagocytosis, (2) coated pits, and (3) direct fusion. Morphological evidence suggests that internalized HIV may escape into the cytoplasm of the target cell by uncoating at the endosomal membrane. Based on CD4 antibody binding and CD4 antibody blocking experiments, HIV entry does not appear to be mediated by a viral CD4 receptor. Productivity of I407 infection was confirmed by virus isolation from cocultured MT-4 lymphocytic cells, reverse transcriptase assay, p24 antigen ELISA, in situ HIV mRNA hybridization, and Southern dot blot analysis. Contrary to infection with free virus, the cell-to-cell infection was not blocked by anti-gp120 or antiviral serum from HIV-positive individuals. It appears that HIV transmission within the confined space between contacting cells enables HIV to evade immune protection provided by neutralizing antibodies. Our results reveal a mechanism of HIV infection of epithelial cells which is triggered by cell-cell contact. Furthermore, these observations offer an insight into the cellular sequence of events which may take place during sexual transmission of HIV across an intact epithelial barrier.

CD4 Antigens↗

PubMed1992

HIV-1 infection of the trophoblast cell line BeWo: a study of virus uptake.

An in vitro model has made it possible to demonstrate HIV transmission from infected lymphocytes to placental trophoblast cells via endocytosis. Upon addition to cultured trophoblast cells (BeWo), chronically HIV-infected lymphocytic cells (MOLT-4) adhered to the epithelial cells via a complex of newly induced microvilli. Though viruses were infrequently seen in the infected lymphocytic cell line, mature virions appeared promptly and profusely in the interstices between the interdigitating microvilli of the two cell types. Virions appeared to bud from the lymphocyte donor cells at the point of cell-to-cell contact and were rapidly taken up by the trophoblast cells via an endocytic mechanism involving coated pits, endosomes, and lysosomes. Electron microscopic observations suggest that HIV may later escape into the trophoblast cytoplasm by fusing with the endosome membrane or by lysing the lysosome membrane. Coincubation for 1 h was sufficient to establish HIV infection in the trophoblast cell line. Four weeks after thoroughly washing out the donor lymphocytic cells, HIV RNA was demonstrated in clusters of BeWo cells by in situ hybridization, and p24 antigen was localized with immunocytochemistry. Soluble CD4 did not block infection as measured by p24 ELISA. The HIV infection was productive and chronic as demonstrated by cocultivating the BeWo cells with indicator lymphocytes 4 weeks after the initial infection. This study, demonstrating a mechanism of HIV transmission, expands upon previous observations that trophoblast cell lines lacking the CD4 viral receptor can nevertheless be infected by HIV and can support productive infection.

CD4 Antigens↗

PubMed1992

Association of mycoplasma with HIV-1 and HTLV-I in human T lymphocytes.

Recent findings from a number of investigators suggest associations between mycoplasma and HIV or AIDS. We used a quantitative morphometric technique to analyze electron micrographs of human T lymphocytes that were infected with both mycoplasma and/or HIV-1. We observed that lymphocytes which were associated with HIV-1 were much more likely to be associated with mycoplasma than cells that were not (p < .001). Similarly, cells with associated HTLV-I were more likely to be associated with mycoplasma than cells which were not associated with mycoplasma (p < .0001). In addition, mycoplasma and virus were observed in the same region in 90% of cases. These observations suggest that adherence of mycoplasma to lymphocytes that are chronically infected with human retrovirus may trigger viral release.

Cell Line↗

PubMed1992

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.

Cell Adhesion↗

PubMed1992

Patient and provider satisfaction with medical care.

BACKGROUND: This study compares patient and provider satisfaction with medical care and waiting time in a large family medicine residency program. Few published studies have dealt with both patient and provider perceptions. METHODS: Telephone interviews were conducted with 156 adult, English-speaking patients who were randomly selected from daily appointment schedules. The patients were asked to rate their satisfaction with 10 aspects of medical care and to estimate the length of time they waited to see their physicians. Sixty-five family health care providers responded to the same survey items through a self-administered questionnaire. RESULTS: In general, 97% of patients and 89% of providers were satisfied with the overall medical care provided at the family health center. Approximately 8% of patients and 22% of providers were dissatisfied with waiting time, and 11% of patients and nearly 60% of providers were dissatisfied with appointment scheduling. Patients' estimates of waiting time for care (mean = 16.1 minutes) were significantly shorter than providers' estimates (mean = 27.5 minutes). Patients who were dissatisfied with the length of waiting time estimated waiting 41.8 minutes, while satisfied patients estimated waiting 13.3 minutes (P less than .001). CONCLUSIONS: Family medicine patients reported higher levels of satisfaction with medical care than did providers. Both groups were the least satisfied with access to care.

Adolescent↗

PubMed1991

Simple colorimetric cell-cell adhesion assay using biotinylated lymphocytes.

A new approach for quantitating lymphocyte adhesion based on labeling the lymphocyte plasma membrane with water-soluble biotin was developed. Adherent biotinylated lymphocytes were quantitated by measuring OD values of a colored substrate representing the amount of bound avidin-peroxidase. The lymphocyte adhesion assay based on the high affinity of avidin to biotin was considerably more sensitive when compared to rose bengal or [3H]thymidine labeling methods. The end-point of sensitivity is approximately 1000 lymphocytes which is clearly an improvement over the rose bengal or radiolabeling techniques with a detection limit of respectively 15 x 10(3) and 7.5 x 10(3) lymphocytes added to wells at the beginning of the assay. The method has the advantage of being rapid and simple and offers an alternative to adhesion assays based on cell ELISAs using cell-specific monoclonal antibodies.

Biotin↗

PubMed1991

Spermiation and sperm maturation in the marmoset.

The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hook-shaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.

Animals↗

PubMed1991

Maturation of the rat cumulus-oocyte complex: structure and function.

The cumulus cells that surround the mammalian oocyte become dispersed following the preovulatory surge of the pituitary gonadotropin, luteinizing hormone (LH). We have examined cumulus-oocyte complexes of PMSG-primed immature rats before and at 1, 2, 3, 4, 6, and 8 hr after injection of human chorionic gonadotropin (hCG), which acts on the rat ovary like the pituitary gonadotropin. Associations between projections of the cumulus cells and the oocyte were analyzed in thin sections. We observed that some cumulus projections were greatly enlarged where they associate with the oocyte. These enlarged regions were filled with numerous small vesicles. Gap junctions between cumulus cell projections and the oocytes were small. We quantitated the number and size of gap junctions between cumulus cells. The number of small gap junctions (less than 1 microM) between cumulus cells did not change significantly over the 8-hr period after hCG administration. Larger gap junctions, however, showed a general downward trend beginning after the third hour post hCG. Light microscopic observations of plastic sections revealed that dispersion of the cumulus oophorus is not observed until after 4 hr post-hCG, but between 4 and 8 hr after gonadotropin administration the cumulus becomes markedly dispersed. In the majority of the oocytes in these complexes the germinal vesicle (GV) displayed some irregularity in shape at 2 hr post-hCG, although absence of the GV was not observed until later. Our observations suggest a new means of communication in the cumulus-oocyte complex by the vesicle-filled enlargements of the cumulus cell projections at the oocyte surface. They further indicate that the decrease in metabolic coupling observed in rat cumulus-oocyte complexes soon after exposure to LH is not associated with a change in number and size of the gap junctions between the cumulus cells. We suggest that it is either the disruption of the gap junctions at the region of contact of the cumulus cell projections with the oocyte surface or the operation of a gating mechanism that blocks the junctional channels without affecting their morphological appearance that is responsible for uncoupling of the oocyte from the cumulus cells.

Animals↗

PubMed1991

Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa.

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.

Animals↗