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Biomedical subjects

M A Stanley

Publications and source records attributed to M A Stanley.

At least 73 records · Page 4Linked to original sources
PubMed1991

Social phobia: Axis I and II correlates.

The presence of Axis I and Axis II disorders in 71 social phobic patients was examined. Generalized anxiety disorder was the most common secondary Axis I disorder, followed by simple phobia. Avoidant personality disorder and obsessive-compulsive personality disorder were the most common Axis II diagnoses, and 88% of the sample exhibited features of these 2 personality styles. Subjects with additional Axis I diagnoses were more anxious and depressed than those with no additional Axis I disorder. Social phobics with additional Axis II disorders were more depressed but not more anxious than those with no Axis II diagnosis. Furthermore, those with an additional Axis I disorder had higher scores on measures of neuroticism, interpersonal sensitivity, and agoraphobia. The prevalence and impact of additional Axis I and II disorders on the etiology, maintenance, and treatment outcome for persons with social phobia are discussed.

Adult↗
PubMed1989

Properties of a non-tumorigenic human cervical keratinocyte cell line.

A human cervical keratinocyte cell line, W12, has been initiated from a low-grade cervical lesion histologically diagnosed as CIN I. This cell line has, to date, undergone over 300 generations in vitro with an average doubling time of 30 hr: an aneuploid karyotype has developed with progressive in vitro growth. W12 contains HPV16 DNA present at approximately 100 copies and the state of the viral DNA over a number of passages has been examined. The HPV16 DNA is stably maintained at high copy number over several passages and restriction enzyme analysis together with electrophoresis of uncleaved viral DNA indicate that it is present predominantly as the episomal molecule. W12 cells exhibit a typical keratinocyte morphology and, when transplanted into the flank of the nude mouse, form an epithelial lesion with the histological features of CIN I/II.

Adult↗
PubMed1989

MHC class II antigen expression is not induced on murine epidermal keratinocytes by interferon-gamma alone or in combination with tumour necrosis factor-alpha.

Epidermal keratinocytes are induced to express MHC class II molecules in a variety of disease states associated with immune activity. To investigate the mechanism of this process we have exposed murine and rat keratinocytes to a variety of lymphokines and monitored changes in their MHC molecule expression. Murine cultured keratinocytes were treated with recombinant interferon-gamma (IFN-gamma), and MHC antigen expression quantified by flow cytometry. IFN pretreatment resulted in the up-regulation of class I molecule expression, but no class II expression was detected. In addition, cultured murine keratinocytes exposed to a combination of recombinant tumour necrosis factor (TNF)-alpha and IFN-gamma, or crude lymphocyte supernatants, failed to show positive membrane staining for class II molecules. However, rat keratinocytes cultured under conditions identical to murine cells were induced to express class II molecules after IFN-gamma pretreatment. The inability of IFN to induce class II expression on murine keratinocytes appears not to result from cell culture, as subcutaneous injection of IFN fails to induce epidermal class II antigen expression. However, class II expression can be induced on rat epidermis in vivo. Thus, the response of epidermal keratinocytes to IFN-gamma appears to show species variation.

Animals↗
PubMed1989

The influence of MHC-compatible and MHC-incompatible antigen-presenting cells on the survival of MHC-compatible cultured murine keratinocyte allografts.

Our group has shown previously that APC-depleted cultured epidermal keratinocytes show prolonged survival when grafted onto normal MHC-incompatible adult mice. We show here that in vitro culture also improves significantly the survival of MHC-compatible keratinocyte allografts, although these nonrejected grafts are repopulated by host cells identified by their dendritic morphology and phenotype (class II+, leukocyte-common antigen+) as APCs. Reconstitution of cultured grafts, immediately prior to transplantation, with MHC-compatible dendritic cells of either donor or recipient origin, results in graft rejection--thus demonstrating that cultured cells can be rejected by the recipient animal--and suggests that a paucity of APCs in the immediate postgrafting period is responsible for the privilege afforded these grafts.

Animals↗
PubMed1989

In vivo and in vitro effects of v-fos and EJ-Ha-ras oncogene expression in murine epidermal keratinocytes.

Primary neonatal Balb/c keratinocyte (NEK) cultures grown, using 3T3 feeder cell support in high calcium, serum supplemented medium, were transfected with EJ-Ha-ras or v-fos DNA sequences and pSV2 neo. Several neo resistant clones were isolated and several established cell lines expressing the transfected gene products derived. Two of these lines, Ras 8 and Fos 1, have been examined in detail with respect to their self renewal capacity and differentiation potential in vitro and in vivo. In vitro, both lines (when compared to normal NEK) have an extended probably immortal phenotype, enhanced colony forming efficiency (a measure of in vitro self renewal capacity) and a reduction in growth factor and serum dependence. When grafted onto syngeneic recipients neither cell line is tumourigenic. Instead a histologically abnormal epithelium with no stratum corneum and with features specific to the oncogene expressed is formed. The extent of the histological atypia correlates with the in vitro alterations in cytoskeletal peptides as revealed by 2D PAGE. However despite the gross histological abnormality there is no alteration in the in vivo self renewal capacity (measured as the number of grafted cells required for epidermal reformation) between normal NEK and the Ras 8 or Fos 1 lines; in each case a minimum of 10(5) cells/1.14 cm2 is required before a full thickness epithelium forms.

Animals↗
PubMed1988

Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

Animals↗
PubMed1987

Prolonged survival of cultured keratinocyte allografts in the nonimmunosuppressed mouse.

The effect of in vitro culture on the survival of allografts of epidermal keratinocytes has been examined using a mouse model. Female BALB/c tail epidermal cells were cultured from single cell suspensions to form confluent sheets that were grafted onto male CBA recipients using a transplantation technique that ensured separation of donor graft from host skin. Animals were killed at defined intervals, and the status of the grafts determined histologically. Full thickness skin allografts rejected at 13-15 days. Allografts of epidermis (obtained by enzymatic cleavage at the dermoepidermal junction) rejected at 19-20 days. Cultured keratinocyte allografts were not rejected at least within 70 days and had a histological appearance identical to syngeneic controls. The expression of MHC class I and class II determinants and the leukocyte common (Ly5) surface marker on the donor cells before and after culture were examined using indirect immunofluorescence and monoclonal antibodies. These and other cytochemical studies showed that freshly dissociated tail epidermal cells contained 0.3% of cells that expressed membrane-bound ATP-ase activity, Ia antigens, and the Ly5 surface antigen. These are the Langerhans' cells of the epidermis. In culture, these cells decrease so that by day 8 of culture, no such cells can be detected. At confluence, there are no Ia positive cells, but all cells express MHC class I antigens and stain with an antikeratin antibody. The loss of Ia expression on culture correlates with a decreased stimulation of the class II H-2d-restricted T cell clone D7.1 by cultured keratinocytes compared with freshly dispersed epidermal cells. Furthermore, cultured keratinocytes fail to prime allogeneic mice as determined by the survival of whole thickness skin grafts, whereas freshly dispersed cells induce an accelerated rejection. The results suggest that the survival of cultured keratinocyte allografts is due to the elimination of cells expressing Ia antigens and supports the passenger leukocyte theory of graft rejection.

Animals↗
PubMed1987

Effects of gamma radiation on spermatogenesis and fertility of male Amblyomma americanum (Acari: Ixodidae).

Amblyomma americanum males were treated with 0.5, 1, 2, 3, 4, 8, and 16 krad of gamma radiation. Testes of ticks treated with 2, 3, 4, 8, and 16 krad were smaller than those of ticks irradiated at lower levels and controls. No recognizable alteration in timing of spermatogenesis was noted among the different radiation groups, but severe breakdown and depletion of germinal cells was noted at 4, 8, and 16 krad. Percent hatch of larvae from crosses of irradiated males and untreated females decreased with increasing radiation level. No hatch was observed from eggs of females mated to males treated at 2 krad or higher.

Animals↗
PubMed1986

Chromosome damage induced by herpes simplex virus type 1 in early infection.

The chromosome damage induced by herpes simplex virus type 1 (HSV-1) in vitro was examined up to 6 h after infection using HT-1080 cells. Initial damage occurring within 3 h was specific, involving uncoiling of chromosome 1q12-21 and to a lesser extent the pericentric regions of chromosomes 9, 16 and satellited chromosomes. For the initial unwinding, synthesis of the immediate early class of HSV proteins needed to occur as was demonstrated using HSV-1 temperature-sensitive mutants tsK and tsB7 and two viral inhibitors, beta-propiolactone and psoralen plus long wavelength u.v. light. Later damage included chromatid breaks, acentric fragments and pulverization which did not take place until synthesis of delayed early proteins had begun.

Cell Line↗
PubMed1985

Responses of human cervical keratinocytes in vitro to tumour promoters and diethylstilboestrol.

We have compared the responses of normal human cervical keratinocytes (HCE) to diethylstilboestrol (DES), and the promoting agents, phorbol-12-myristate-13-acetate (PMA) and mezerein using the loss of cloning efficiency as a measure of terminal differentiation in vitro. Dose-response studies showed that normal HCE are growth inhibited by chronic exposure to DES at concentrations greater than or equal to 2.5 X 10(-5) M, to PMA at concentrations greater than 10(-8) M and mezerein at concentrations greater than 10(-9) M. Compared to acetone controls, promoter or DES-treated cells exhibited a 10- to 12-fold increase in cornified-envelope formation. Normal HCE exhibit a heterogeneous response to PMA in that 85-90% of colony-forming cells lose their colony-forming ability after a 24-h exposure to 10(-6) M PMA. The PMA-resistant subpopulation, PMAR, remains constant and is not reduced even after 96 h chronic exposure to PMA. In contrast, the colony-forming ability of normal HCE is almost totally suppressed after 24 h exposure to 10(-6) M mezerein. After 24 h incubation with 5 X 10(-5) M DES, 20% of normal HCE are capable of colony formation but this resistant fraction is eliminated after 96 h chronic exposure. Cornified-envelope formation was negligible in malignant cervical keratinocytes grown in the presence of DES or promotors and these cells were characterised by a very large PMAR fraction - 85 - 90% of cells retained colony-forming ability after exposure to 10(-6) M PMA for 24 h. Furthermore, 90-100% of malignant cervical keratinocytes retained their colony-forming capacity after exposure to 10(-6) M mezerein. However, colony-forming ability declined steadily in the presence of 5 X 10(-5) M DES and after 96 h only a tiny fraction, 1% of malignant cervical keratinocytes could form colonies on replating. The mechanisms by which DES inhibits growth and induces cornified-envelope formation in HCE would appear to be distinct from those activated by PMA and mezerein.

Carcinogens↗
PubMed1984

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells.

The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5 X 10(3)/cm2 in the presence of 2 X 10(4)/cm2 lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10(-10) M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size. Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.

Cell Division↗
PubMed1984

The effect of diethylstilboestrol on the in vitro growth of human ectocervical cells.

The effect of chronic exposure to diethylstilboestrol (DES) over the concentration range 10(-4)-10(-6) M on the in vitro growth of cells derived from the squamo -columnar junction of the human cervix has been examined. DES over the dose range 10(-4)-2 X 10(-5) M inhibits cervical epithelial cell (HCE) growth in terms of colony formation and colony expansion. This effect involves both an effect on cell proliferation, as measured by cell number and mitotic index, and an induction of differentiation as measured by an increase in the number of PAS positive cells (glycogen accumulating cells) and in the number of cells with cornified envelopes. At lower doses of DES, 10(-5)-5 X 10(-6) M, the efficiency of colony formation and colony size are not inhibited but mitotic index and cell number are decreased and the fraction of differentiating cells is increased. The effects of DES on HCE cell growth can be modified to some extent by epidermal growth factor (EGF). EGF at 10 ng/ml, increases the fraction of proliferating cells, induces cellular hypertrophy, reduces stratification and the fraction of differentiating cells in HCE colonies. DES, at all concentrations other than 10(-6) M, in the presence of EGF inhibits cell proliferation but does not prevent the EGF induced cellular hypertrophy, reduction in stratification or reduction in the fraction of differentiating cells.

Cell Differentiation↗
PubMed1984

Immunofluorescent studies of human cervical epithelia in vivo and in vitro using antibodies against specific keratin components.

The human cervix consists of a vaginal ectocervix covered by stratified squamous epithelium and a uterine endocervix lined by simple mucous secreting epithelium. In this study, changes in keratin polypeptide composition in ectocervical and endocervical epithelia in vivo and in vitro have been investigated. Immunofluorescent studies wer done using a polyclonal antibody raised against a 40 kd keratin polypeptide (anti-40 kd) and a monoclonal antibody (LE61) which recognizes a keratin polypeptide of simple epithelia. In sections of ectocervix, anti-40 kd staining was restricted to the basal layer of the epithelium. In sections of endocervix all epithelial cells were positively stained by anti-40 kd and LE61. Regions of squamous metaplasia were negative for LE61 and weakly positive for anti-40 kd. Cultured ectocervical epithelial cells were negative for LE61 but some cells around the periphery of colonies were positive using anti-40 kd. Cultured endocervical epithelial cells stained positively with LE61 and anti-40 kd. Both antibodies stained a cytoplasmic filamentous network and double staining revealed a codistribution of the labelled filaments. After around 14 days in culture endocervical epithelial cells underwent a morphological change and assumed a keratinocyte appearance suggesting an in vitro equivalent of squamous metaplasia. However, such cells retain their LE61 antigenicity.

Antibody Specificity↗
PubMed1979

Growth requirements of human cervical epithelial cells in culture.

Diploid cells derived from the portio surface of the cervix uteri of adult women can be grown in culture, from single cells. Ultrastructurally the colonies of growing cells show the features of stratified squamous epithelium and exhibit some of the differentiated characteristics of this epithelium in vivo. The successful serial cultivation of cervical epithelial cells, HCE, depends upon the presence of a non-dividing fibroblast feeder layer which provides both attachment and growth factors for epithelial cells. Lethally irradiated Wi-38, cervical fibroblasts or mouse fibroblast lines are all effective in promoting HCE colony formation and growth. Hydrocortisone and epidermal growth factor increase HCE colony size in the presence of a feeder cell layer. Culture lifetime was studied in five strains and ranged from 20-38 population doublings, with cells from older patients exhibiting a shorter culture life-time.

Adult↗