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D Sheppard

Publications and source records attributed to D Sheppard.

At least 55 records · Page 3Linked to original sources
PubMed1998

The integrin alphavbeta6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin.

The integrin alphavbeta6 is expressed on a variety of epithelial cells during dynamic processes including organogenesis, tissue injury and malignant transformation. However, because of the lack of tools to specifically inhibit the function of this integrin, little is known about its effects on cell behavior. To directly examine the role of this integrin in cell migration, we used keratinocytes derived from wild-type mice or mice expressing a null mutation in the beta6 subunit (beta6-/-) to perform migration assays in vitro. Migration on the known alphavbeta6 ligand, fibronectin was reduced in keratinocytes from beta6-/- mice. Interestingly, keratinocytes from beta6-/- mice also demonstrated markedly reduced migration on vitronectin, a protein not previously known to be a ligand for alphavbeta6. An anti-alphavbeta6 monoclonal antibody 10D5, generated by immunization of beta6-/- mice with murine keratinocytes, inhibited adhesion and migration of wild-type keratinocyte on both vitronectin and fibronectin to levels similar to those seen with keratinocytes from beta6-/- mice. alphavbeta6-mediated migration on both ligands was dramatically augmented by treatment with phorbol myrisate acetate (PMA) or with hepatocyte growth factor, and augmentation of migration by either stimulus could be abolished by the PKC inhibitor GF109203X, suggesting a critical role for PKC in enhancement of alphavbeta6-mediated cell migration.

Animals↗
PubMed1998

Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice.

BACKGROUND: The pathogenesis of asthma is believed to reflect antigen-induced airway inflammation leading to the recruitment of eosinophils and activation of mast cells through cell-associated IgE. Controversies persist however, regarding the relative importance of different pathogenic cells and effector molecules. MATERIALS AND METHODS: A variety of gene-targeted mice were examined for the induction of cholinergic airway hyperresponsiveness (AH), allergic airway inflammation, mucus production, and serum IgE reactivity following intratracheal challenge with a potent allergen. AH was determined using whole-body plethysmography following acetylcholine challenge. Where possible, results were confirmed using neutralizing antibodies and cell-specific reconstitution of immune deficient mice. RESULTS: T and B cell-deficient, recombinase-activating-gene-deficient mice (RAG -/-) failed to develop significant allergic inflammation and AH following allergen challenge. Reconstitution of RAG -/- mice with CD4+ T cells alone was sufficient to restore allergen-induced AH, allergic inflammation, and goblet cell hyperplasia, but not IgE reactivity. Sensitized B cell-deficient mice also developed airway hyperreactivity and lung inflammation comparable to that of wild-type animals, confirming that antibodies were dispensable. Treatment with neutralizing anti-IL-4 antibody or sensitization of IL-4-deficient mice resulted in loss of airway hyperreactivity, whereas treatment with anti-IL-5 antibody or sensitization of IL-5-deficient mice had no effect. CONCLUSIONS: In mice, CD4+ T cells are alone sufficient to mediate many of the pathognomonic changes that occur in human asthma by a mechanism dependent upon IL-4, but independent of IL-5, IgE, or both. Clarification of the role played by CD4+ T cells is likely to stimulate important therapeutic advances in treatment of asthma.

Adoptive Transfer↗
PubMed1997

Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells.

Viral entry into host cells depends upon specific interactions between virus attachment proteins and cell surface receptors that enable virus binding and internalization of virus and/or the virus-receptor complex. We have recently reported that the ubiquitous cell surface molecule, decay-accelerating factor (DAF), is a major cell attachment receptor for Coxsackieviruses B1, B3, and B5. However, DAF permits only virus binding and not virus internalization, invoking the presence of secondary or accessory receptors. Among the known receptors for enteroviruses are members of the cell adhesion molecule family known as integrins. In the present study, we found that expression of the epithelial-restricted integrin, alpha v beta 6, on colonic epithelial cells significantly enhanced Coxsackievirus B1-mediated cell lysis. Importantly, the viral-mediated cell killing required the presence of the 11-amino-acid C-terminal cytoplasmic extension unique to the beta 6 subunit, providing the first evidence of regulation of viral infectivity by integrin cytoplasmic domains. These results indicate that alpha v beta 6 expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be essential in the conversion of asymptomatic enterovirus infection into clinically apparent disease.

Antigens, Neoplasm↗
PubMed1997

Stromal fibroblasts influence oral squamous-cell carcinoma cell interactions with tenascin-C.

In this study we identified tenascin-C (TN-C) and one of its integrin receptors, alpha(v)beta6, in oral squamous-cell carcinoma (SCC) specimens. Neither TN-C nor alpha(v)beta6 are expressed in normal oral mucosa. We also studied 2 human oral squamous-cell carcinoma cell lines: the highly invasive HSC-3 cells, and the poorly invasive SCC-25 cells. We determined that adhesion of these cells to TN-C involves both alpha2 and alpha(v) integrins. Migration on TN-C by oral SCC cells required fibroblast-conditioned medium and did not occur in its absence. This migration was blocked by anti-alpha2 and anti-alpha(v) antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor-beta1. When seeded on TN-C, the poorly invasive SCC-25 cells formed alpha(v)beta6-positive focal contacts; the HSC-3 cells did not. HSC-3, SCC-25 and PTF cells secrete TN-C into the culture medium, as determined by Western blot. However, when HSC-3 cells were inoculated into the floor of the mouth of nude mice, only murine TN-C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC-3 cells do not secrete TN-C. Our results demonstrate that alpha(v)beta6 and tenascin-C are neo-expressed in oral squamous-cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior.

Animals↗
PubMed1997

Renal artery stent placement in renal artery stenosis: technical and early clinical results.

We report the technical and early clinical results of renal artery stent placement in 29 consecutive patients treated at a single centre over a 30-month period, employing the Palmaz balloon-expandable stent. Of 32 arteries treated, 23 (72%) were atheromatous, ostial stenoses. Immediate technical success was achieved in all 29 patients. Follow-up angiography was performed on 25 patients at 6.7 months (mean) and demonstrated a patient restenosis rate of 16%. All surviving patients were followed up for a minimum of 6 months. Blood pressure control was improved in eight (50%) of hypertensive patients, and renal function improved in seven (33%) and stabilized in six (29%) patients with chronic renal impairment (serum creatinine > 150 mumols/l). Complications occurred in seven (24%) of patients, including one procedure-related death. Our experience indicates that stent placement has an initial high technical success rate in renal artery stenosis and that this patency is maintained at repeat angiography with a low restenosis rate. Renal artery stenting is likely to extend the role of percutaneous renal revascularization especially in atheromatous ostial lesions. A randomized trial will be required to evaluate its role compared with balloon angioplasty.

Adult↗
PubMed1997

Informed consent issues in home care.

Providers have a responsibility to inform patients about the care they receive. With this responsibility, however, come liabilities. Agencies need to be aware of both the responsibilities and liabilities involved with informed consent and address them in everyday operations--failure to comply with the federal requirements can result in termination or nonrenewal of a provider's Medicare agreement.

Advance Directives↗
PubMed1996

Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

Animals↗
PubMed1996

Identification of a sequence within the integrin beta6 subunit cytoplasmic domain that is required to support the specific effect of alphavbeta6 on proliferation in three-dimensional culture.

The integrin alphavbeta6 augments the proliferation of epithelial cells in collagen gels and in vivo. This effect depends on the presence of a unique carboxyl-terminal region of the beta6 subunit cytoplasmic domain. In the present study, we have utilized deletional and alanine substitution mutagenesis within this region to map the amino acids responsible for alphavbeta6-mediated proliferation in more detail. Replacement or deletion of any of 6 amino acids (glutamic acid 778, lysine 779, lysine 781, valine 782, aspartic acid 783, and leucine 784) largely abolished the proliferative effects of alphavbeta6, but none of the mutants examined interfered with alphavbeta6-mediated cell adhesion or with localization of alphavbeta6 to focal adhesions. These findings suggest that residues contained within the sequence EKXKVDL are critical for the effects of alphavbeta6 on proliferation in collagen gels and that pathways initiated by interaction with this sequence are distinct from those required for integrin-mediated cell attachment or focal adhesion formation.

Alanine↗
PubMed1996

Differential effects of the integrins alpha9beta1, alphavbeta3, and alphavbeta6 on cell proliferative responses to tenascin. Roles of the beta subunit extracellular and cytoplasmic domains.

Members of the integrin family manifest considerable overlap in ligand specificity, and many cells have the capacity to express multiple integrin receptors for the same ligand. For example, at least 5 different integrins recognize tenascin as a ligand, and 4 of these bind to the same region of the protein, the third fibronectin type III repeat (TNfn3). We utilized colon carcinoma cells (SW480) that do not normally attach to TNfn3 to examine the possibility that ligation of different integrin receptors for this ligand would induce different effects on cell behavior and intracellular signaling. Heterologous expression of the tenascin receptors alphavbeta3 and alpha9beta1 produced comparable effects on cell adhesion and spreading on TNfn3, but alphavbeta3-transfectants proliferated considerably better on each concentration examined. alphavbeta6-transfectants attached (although less avidly), but completely failed to spread or proliferate. Expression of a chimeric beta subunit composed of the beta3 extracellular domain fused to the beta6 transmembrane and cytoplasmic domains resulted in adhesion and spreading similar to that seen with beta3-transfectants, but considerably less proliferation. When the same cell lines were plated on fibronectin, alphavbeta6-transfectants spread and proliferated as well as cells transfected with the chimeric beta3/beta6 subunit, but, again, neither cell line proliferated as well as cells expressing alphavbeta3. Cell proliferation was always associated with spreading and with phosphorylation of the focal adhesion kinase, paxillin, and the mitogen-activated kinase, Erk2, but cell attachment in the absence of spreading or proliferation was not associated with phosphorylation of any of these proteins. These data suggest that different integrin receptors for a single ligand can produce markedly different effects on cell proliferation, and that both the extracellular and cytoplasmic domains of integrin beta subunits contribute to these differences.

Antigens, CD↗
PubMed1996

Epithelial integrins.

The integrin family was originally described as a family of adhesion receptors, utilized by cells for attachment to and migration across components of the extracellular matrix. Epithelial cells in adult tissues are generally stationary cells, but these cells nevertheless express several different integrins. This review will discuss the evidence that integrins on epithelial cells are also likely to function as signaling molecules, allowing these cells to detect attachment or detachment, and changes in the local composition of ligands. Signals initiated by integrins appear to modulate epithelial cell differentiation, proliferation, survival, and gene expression. Because the local concentration of integrin ligands is altered by injury, inflammation, and remodeling, signals initiated through integrins are likely to play important roles in the responses of epithelial cells to each of these processes.

Adult↗
PubMed1996

Multiplicity of fibronectin-binding alpha V integrin receptors in colorectal cancer.

Current data from in vitro and in vivo animal models indicate that fibronectin-binding integrin receptors expressed by colon cancer cells may regulate tumour growth. While individual members of the beta 1 subfamily of integrins have now been clearly identified in colorectal cancer, little information exists with respect to the alpha V subfamily. In the present study we show that alpha V can associate with multiple and different beta subunits capable of binding fibronectin in this tumour type. This is likely to have functional implications for growth and spread of colorectal cancer.

Antigens, CD↗
PubMed1996

Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin.

The integrin alpha v beta 6 is only expressed in epithelial cells. In healthy adult epithelia, this receptor is barely detectable, but expression is rapidly induced following epithelial injury. Mice homozygous for a null mutation in the gene encoding the beta 6 subunit had juvenile baldness associated with infiltration of macrophages into the skin, and accumulated activated lymphocytes around conducting airways in the lungs. Beta 6-/- mice also demonstrated airway hyperresponsiveness to acetylcholine, a hallmark feature of asthma. These results suggest that the epithelial integrin alpha v beta 6 participates in the modulation of epithelial inflammation. Genetic or acquired alterations in this integrin could thus contribute to the development of inflammatory diseases of epithelial organs, such as the lungs and skin.

Alopecia↗
PubMed1996

Keratinocytes in human wounds express alpha v beta 6 integrin.

Cell adhesion receptors of the integrin family play a major role during re-epithelialization of human wounds. We have previously documented that the expression of alpha v family integrins is induced in keratinocytes of mucosal wounds [1]. In the present investigation, we extended these studies to determine whether alpha v beta 6 integrin is expressed during wound healing in humans. Mucosal and epidermal wound sections from 1- to 7-day-old wounds were used for immunolocalization of integrins and their putative ligands. In addition, freshly isolated epidermal keratinocytes were used to study integrin expression in vitro. Expression of alpha v beta 6 integrin appeared relatively late during mucosal and dermal wound healing. Maximal expression was seen in 7-day-old wounds in which epithelial sheets had fused and granulation tissue was present. Fibronectin and tenascin, both possible ligands for alpha v beta 6 integrin, were found concentrated underneath the basal epithelial cells expressing this receptor, and the maximal expression of tenascin coincided with that of alpha v beta 6 integrin. Freshly isolated epidermal keratinocytes did not stain for alpha v beta 6 integrin but began to express this integrin after subculturing. Our results suggest that the expression of alpha v beta 6 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing.

Antigens, Neoplasm↗
PubMed1996

Fibronectin and its alpha 5 beta 1-integrin receptor are involved in the wound-repair process of airway epithelium.

The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.

Antibodies↗
PubMed1996

Differential regulation of airway epithelial integrins by growth factors.

The pattern of integrin expression on human airway epithelium changes significantly in injury or inflammation. In particular, two integrins, the fibronectin receptor, alpha 5 beta 1 and the fibronectin/tenascin receptor alpha v beta 6, are expressed at low or undetectable levels in normal airways in vivo but are induced in response to airway epithelial injury. We investigated the effects of various growth factors known to be present in the airways on the expression of constitutively expressed and inducible airway epithelial integrins using flow cytometry. In primary cultures of human airway epithelial cells, transforming growth factor-beta 1 (TGF beta 1) dramatically increased expression of alpha v beta 6 and essentially did not affect the expression of any other integrin, including alpha 5 beta 1. In contrast, epidermal growth factor (EGF) upregulated surface levels of both alpha v beta 6 and alpha 5 beta 1. Together, TGF beta 1 and EGF had an additive effect on alpha v beta 6 and alpha 5 beta 1 expression while increasing levels of alpha 2 beta 1 and decreasing expression of alpha 3 beta 1- and alpha 6-containing integrins. In contrast, the transformed airway epithelial cell line, BEAS-2B, expressed a markedly different repertoire of integrins. Integrin expression on BEAS-2B cells was not affected by any of the growth factors tested in this study. These results demonstrate that, in primary cultures of human airway epithelial cells, the pattern of integrin expression can be dramatically altered by growth factors. The inducible integrins, alpha v beta 6, and alpha 5 beta 1 are most subject to regulation by growth factors and expression of each of these can be differentially regulated. The differential regulation of the two principal fibronectin receptors on airway epithelial cells suggests that they may mediate different cellular responses to fibronectin.

Antibodies, Monoclonal↗
PubMed1996

Colour Doppler ultrasound in renal artery stenosis: intrarenal waveform analysis.

Renal artery stenosis (RAS) is the commonest secondary cause of hypertension and may result in renal ischaemia with resultant renal failure. Recent studies hve suggested that colour Doppler ultrasound, with spectral analysis of the intrarenal waveforms, can identify those patients with a significant RAS. A prospective study was performed in which colour Doppler ultrasound was compared with angiography in 73 patients (143 kidneys) presenting for renal angiography. Colour Doppler ultrasound was unsuccessful in 16% of kidneys due to a combination of technical failures and small kidney size. Accessory renal vessels were present in 14% of kidneys on angiography but none was detected by ultrasound. Of the 120 kidneys that had both examinations, no significant difference in intrarenal pulsatility or resistive index was noted between the angiographically stenosed and normal arteries. There were significant differences for intrarenal peak and end diastolic velocities, and acceleration time and index. Of these measurements, acceleration time was the best indicator of RAS. The probability of detecting a high grade RAS in an individual patient did not reach 90% until the acceleration time was prolonged to more than 0.12 s. Intrarenal colour Doppler ultrasound is not a general screening test for RAS and it should be reserved for selected patient groups where the incidence of disease is high. Patients with prolonged acceleration times of more than 0.12 s have a high likelihood of at least 70% RAS and should proceed directly to angiography.

Adult↗
PubMed1996

Distinct structural requirements for interaction of the integrins alpha 5 beta 1, alpha v beta 5, and alpha v beta 6 with the central cell binding domain in fibronectin.

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, the structural requirements for binding to fibronectin differ. In the present study, we report that two additional integrins, alpha v beta 6, and alpha v beta 5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments, alpha v beta 6, like alpha v beta 3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study, alpha v beta 5 only minimally supported adhesion to intact fibronectin, but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat, alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by alpha v beta 5, as well as adhesion mediated by alpha v beta 6. alpha v beta 5 and alpha v beta 6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.

Antigens, Neoplasm↗