Biomedical subjects

C C Hughes

Publications and source records attributed to C C Hughes.

At least 19 recordsLinked to original sources

PubMed2005

Virus infection in remnant native bunchgrasses from invaded California grasslands.

This study examined the effects of infection with barley and cereal yellow dwarf viruses (BYDVs) on wild grass species in California, a region in which native perennial bunchgrasses have been largely replaced by exotic annual grasses. We sought to determine whether these widespread viruses compromise the fitness of wild hosts and thus have the potential to influence grassland dynamics. Plant viruses have been long overlooked in ecological studies, and their influence on wild hosts has often been assumed to be minimal. We examined the short-term and long-term consequences of infection on field-grown individuals from 18 different populations of wild California grasses (from seven native and one exotic species). Barley yellow dwarf virus infection was aggressive in most hosts and markedly impaired host fitness by reducing growth, survivorship, and fecundity. Previous work indicates that the presence of exotic grasses can more than double BYDV incidence in natives. Given the ubiquity of BYDVs, our results suggest that apparent competition and other virus-mediated processes may influence interactions among native and exotic grasses and potentially contribute to shifts in grassland community composition.

California↗

PubMed2001

Endothelial cell costimulation of T cell activation through CD58-CD2 interactions involves lipid raft aggregation.

Human endothelial cells (EC) costimulate CD4(+) memory T cell activation through CD58-CD2 interactions. In this study we tested the hypothesis that EC activate distinct costimulatory pathways in T cells that target specific transcription factors. AP-1, composed of fos and jun proteins, is a critical effector of TCR signaling and binds several sites in the IL-2 promoter. EC augment c-fos promoter activity in T cells; however, deletion analysis reveals no transcription factor binding sites in the promoter uniquely responsive to EC costimulation. Overexpression of AP-1 proteins in T cells augments the activity of an AP-1-luciferase reporter gene equally in the absence or the presence of EC costimulation. Interestingly, EC stimulate a similar 2- to 3-fold up-regulation of AP-1, NF-AT, NF-kappaB, and NF-IL-2-luciferase reporters. CD2 mAbs completely block EC effects on all of these pathways, as well as costimulation of IL-2 secretion. We conclude that EC costimulation through CD2 does not trigger a single distinct costimulatory pathway in T cells, but rather, it amplifies several pathways downstream of the TCR. Indeed, we find that early EC costimulation acts "upstream" of the TCR by promoting lipid raft aggregation, thus amplifying TCR signaling. Soluble CD2 mAbs block EC-induced raft aggregation, whereas cross-linking CD2 promotes aggregation. These data are consistent with the critical role of CD2 in organizing the T cell-APC contact zone.

CD2 Antigens↗

PubMed2000

The basic helix-loop-helix transcription factor HESR1 regulates endothelial cell tube formation.

Human endothelial cells can be induced to form capillary-like tubular networks in collagen gels. We have used this in vitro model and representational difference analysis to identify genes involved in the formation of new blood vessels. HESR1 (HEY-1/HRT-1/CHF-2/gridlock), a basic helix-loop-helix protein related to the hairy/enhancer of split/HES family, is absent in migrating and proliferating cultures of endothelial cells but is rapidly induced during capillary-like network formation. HESR1 is detectable in all adult tissues and at high levels in well vascularized organs such as heart and brain. Its expression is also enriched in aorta and purified capillaries. Overexpression of HESR1 in endothelial cells down-regulates vascular endothelial cell growth factor receptor-2 (VEGFR2) mRNA levels and blocks proliferation, migration, and network formation. Interestingly, reduction of expression of HESR1 by antisense oligonucleotides also blocks endothelial cell network formation in vitro. Finally, HESR1 expression is altered in several breast, lung, and kidney tumors. These data are consistent with a temporal model for HESR1 action where down-regulation at the initiation of new vessel budding is required to allow VEGFR2-mediated migration and proliferation, but re-expression of HESR1 is necessary for induction of tubular network formation and continued maintenance of the mature, quiescent vessel.

Amino Acid Motifs↗

PubMed2000

In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse.

We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.

Animals↗

PubMed2000

Expression of IFN-inducible T cell alpha chemoattractant by human endothelial cells is cyclosporin A-resistant and promotes T cell adhesion: implications for cyclosporin A-resistant immune inflammation.

IFN-inducible T cell alpha chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-gamma-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-gamma-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-alpha does not induce I-TAC mRNA alone, but does act synergistically with IFN-gamma. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.

Capillaries↗

PubMed2000

Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine-human xenograft model.

OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.

Animals↗

PubMed1999

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.

Antigen Presentation↗

PubMed1999

Immunohistochemical analysis, human papillomavirus DNA detection, hormonal manipulation, and exogenous gene expression of normal and dysplastic human cervical epithelium in severe combined immunodeficiency mice.

The cervical squamocolumnar junction of normal and dysplastic human xenografts was maintained in SCID-beige mice. Dysplastic tissue maintained a dysplastic morphology, irregular pattern of keratin expression, elevated levels of cellular proliferation, and human papillomavirus type 16 and/or type 18 DNA. Hyperplastic changes of normal xenografts occurred via high-dose estrogen exposure, and through recombinant adenovirus infection, the introduction and stable expression of an exogenous gene was accomplished.

Adenoviridae↗

PubMed1998

Activation of the CD2 pathway in lamina propria T cells up-regulates functionally active AP-1 binding to the IL-2 promoter, resulting in messenger RNA transcription and IL-2 secretion.

The aim of this study was to identify molecular mechanisms involved in transcriptional regulation of IL-2 expression following CD2 and CD3 activation in lamina propria (LP) T cells. Studies used T cells from normal, ulcerative colitis, and Crohn's disease mucosa and freshly isolated PBMC, PBMC stimulated with IL-2 alone, and PBMC stimulated with IL-2 and cocultured with B cell lines (LP-like T cells). Electrophoretic mobility shift assays were performed with nuclear extracts from cells activated with either anti-CD2 or anti-CD3 Abs. CD2 signaling in LPMC and LP-like T cells led to a pattern of sustained up-regulation of AP-1-binding complexes, whereas CD3 activation resulted in only transient up-regulation. While the pattern of regulation of AP-1 binding observed in normal, uninflamed, or inflamed Crohn's disease LPMC is similar, differences in intensity of AP-1 binding were observed. Activation of LP-like T cells mimics the up-regulation of AP-1 with a kinetic profile similar to that observed with freshly isolated LPMC from Crohn's disease-inflamed tissue. The AP-1 complex formed following CD2 activation is composed of jun/fos heterodimers. The CD2-enhanced responsiveness is reflected in functional analysis experiments utilizing transfection of both multimeric-TRE or IL-2 promoter-luciferase constructs directly into normal, ulcerative colitis, or Crohn's disease LPMC. Our data suggest that activation of LP T cells from normal, ulcerative colitis, or Crohn's disease mucosa through the CD2 pathway leads to induction of AP-1 complexes that bind to the IL-2 promoter, and may play a pivotal role in modulating IL-2 production in the gut.

Animals↗

PubMed1998

Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin.

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.

Animals↗

PubMed1998

Cultures in conflict: a challenge to faculty of academic health centers.

Academic health centers (AHCs) are experiencing turmoil in all three of their traditional missions of teaching, research, and patient care. The authors examine origins of universities and medical education to place in historical context the stresses affecting AHCs at the end of the 20th century. They describe the cultures of the university to suggest strategies for successful adaptation to these stresses. Clashes of values and norms of the cultures within universities and AHCs can hinder effective adaptation to external change. Administrators, researchers, teachers, and clinicians can have strongly conflicting perspectives. For example, business skill is of increasing importance to the survival of the clinical enterprise, but not typically valued by faculty members. University faculty have often considered accountability as antithetical to academic freedom, and, until recently, accountability was not strongly demanded of AHCs. The authors conclude that AHC faculty must transcend the outdated view that the roles of the scholar, scientist, and healer are in opposition to those of the leader and manager. If AHCs are to survive and prosper through their current cultural transition, their faculty must understand all these roles as part of their intellectual and organizational responsibility.

Academic Medical Centers↗

PubMed1997

Consistent transient transfection of DNA into non-transformed human and murine T-lymphocytes.

The ability to analyze transcriptional regulation in non-transformed T-cells has been hampered by the inability to reproducibly transiently transfect these cells with DNA constructs. We have previously demonstrated that normal human whole mononuclear and CD4 T-cells can be consistently transiently transfected with plasmid DNA. Human cells were most receptive to plasmid DNA uptake between 19.5 and 20 h after prestimulation with a submitogenic dose of the polyclonal T-cell activator, PHA. Here we report an alteration and optimization of this protocol for non-transformed murine splenic T-cells, using concanavalin A instead of PHA as the preactivation stimulus. When coupled with the high sensitivity of luciferase reporter gene constructs, this protocol facilitates the analysis of a variety of T-cell-specific promoters in non-transformed T-cells. In addition, we directly demonstrate that murine T-cells are specifically transiently transfected among a population of whole mononuclear cells by using an expression vector for green fluorescent protein.

Animals↗

PubMed1997

c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation.

The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.

Animals↗

PubMed1997

Blockade of CD2-LFA-3 interactions protects human skin allografts in immunodeficient mouse/human chimeras.

A human skin allograft injury model in immunodeficient mice, engrafted with human peripheral blood mononuclear cells from a different donor, has been used to test whether reagents that block human T cell CD2 interactions with its principal ligand, LFA-3 (CD58), can inhibit immune reactions in vivo. In this model, human skin grafts show a reproducible pattern of progressive human T-cell infiltration and human graft microvascular injury that resembles human first-set skin graft rejection. Murine Mab to human LFA-3 or human LFA-3-IgG1 fusion protein, but not isotype-matched control antibodies, each markedly protected skin grafts from leukocyte infiltration and injury. These data provide the first evidence that LFA-3 functions in vivo and establish the ability of this new model to test human-specific immune modulators.

Animals↗

PubMed1996

Porcine endothelial CD86 is a major costimulator of xenogeneic human T cells: cloning, sequencing, and functional expression in human endothelial cells.

Porcine aortic endothelial cells (PAECs), unlike human endothelial cells, express a surface protein recognized by human CTLA4Ig fusion protein that costimulates human T cells through CD28. We have cloned porcine CD86 (pCD86) from an immortalized porcine endothelial cell line, PEC-A, that expresses high levels of this CTLA-4-binding protein. pCD86 mRNA is expressed in PEC-A and PAECs but not in human endothelial cells. Expression of stably transfected pCD86 in CHO cells modestly costimulates human T cell proliferation and IL-2 secretion. Expression of transiently transfected pCD86 in human umbilical vein endothelial cells strongly costimulates IL-2 production by human T cells, comparable to costimulation by PAECs. Costimulation of human T cells by pCD86 in both systems is as effective as costimulation by human CD80 or CD86, and can be blocked by human CTLA4Ig. We conclude that pCD86 contributes to the strong xenoreactivity of porcine endothelium.

Abatacept↗

PubMed1996

IL-4 and IL-13 activate the JAK2 tyrosine kinase and Stat6 in cultured human vascular endothelial cells through a common pathway that does not involve the gamma c chain.

IL-4 and IL-13 each act on human endothelial cells (ECs) to induce expression of vascular cell adhesion molecule-1. On hematopoietic cells. IL-4 responses may be mediated either through a pathway involving gc, the common signaling subunit of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, or through a gc-independent pathway that may be alternatively activated by IL-13. We find that human ECs do not express gc, as detected by indirect immunofluorescence and FACS analysis or by a reverse transcription-PCR method. Like IL-4, IL-13 activates a protein tyrosine kinase that phosphorylates the IL-4R binding protein. In addition, we find that IL-4 and IL-13 each induce tyrosine phosphorylation of the JAK2 tyrosine kinase. Furthermore, both IL-4 and IL-13 induce binding of the Stat6 transcription factor to a consensus sequence oligonucleotide. We conclude that the IL-4 response of human ECs involves the IL-13 shared pathway that is independent of gc, and uses JAK2-Stat6 signaling.

Base Sequence↗

PubMed1996

Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells.

To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter.

B-Lymphocytes↗

PubMed1996

Endothelial cells augment the expression of CD40 ligand on newly activated human CD4+ T cells through a CD2/LFA-3 signaling pathway.

Cultured human endothelial cells (EC) increase CD40 ligand expression on polyclonally activated human peripheral blood CD4+ helper T cells compared to T cells activated in the absence of accessory cells or in the presence of peripheral blood adherent cells or B cells. Induction of CD40 ligand expression appears to be biphasic with early induction observable at 6 h and later induction at 24 h. EC cause T cells to increase CD40 ligand expression during the early phase at 6 h after activation. CD40 ligand expression is restricted to the CD4+ helper T cell subset of the peripheral blood T cells, even when EC is present. Blocking monoclonal antibodies to co-stimulatory molecules on EC and T cells indicate that the CD2/LFA-3 pathway, which also contributes to induction of augmented interleukin-2 (IL-2) secretion is involved in EC-induced up-regulation of CD40 ligand. Exogenous IL-2 can also increase CD40 ligand expression. However, increased IL-2 secretion in the presence of EC can not fully account for endothelial-induced CD40 ligand up-regulation as (1) the effect of exogenous IL-2 is greater at 24 h than at 6 h, whereas the opposite is true for EC; (2) the effect of saturating levels of IL-2 is considerably smaller than that of EC; and (3) blocking of IL-2 receptors does not fully inhibit endothelial effects on CD40 ligand expression. We conclude that EC provide unique co-stimulatory signals that effect the phenotype of activated CD4+ T cells.

Adult↗