IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses. METHODS: Eosinophil mRNA expression of TSLPR and IL-7Rα was examined by real-time quantitative PCR of human eosinophils treated with TNFα and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNFα and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation. RESULTS: Eosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNFα and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNFα and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation. CONCLUSIONS: This study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.

Sequence of Pelvic Examination Does Not Affect Patients With Baseline Vulvovaginal Syndromes: A Randomized Clinical Trial.

OBJECTIVE: The purpose of this study is to determine the optimal sequence in performing a pelvic examination to reduce discomfort in patients with baseline vaginal pain. METHODS: A randomized controlled trial of women presenting for a new appointment at the Drexel Vaginitis Center was conducted. Women were assigned to either group A, a Q-tip touch test, speculum examination, then bimanual examination, or group B, a Q-tip touch test, bimanual examination, then speculum examination. The primary outcome was visual analog scales to assess pain at baseline and after each portion of the examination. Secondary outcomes were responses to questionnaires for self-esteem, quality of life, and sexual function. RESULTS: Two hundred women were enrolled in the trial. For both group A and group B, each portion of the examination was similarly scored regardless of whether the speculum examination was performed before or after bimanual examination. Pain during the speculum examination was higher than pain during the other components of the examination, although not significant (P = 0.65).When looking at reported pain outcomes, outcomes did not differ as a whole or between groups in relation to sexual activity, sexual orientation, and previous hysterectomy. The data were not significantly different between groups for self-esteem scores, sexual dysfunction, or quality of life scores. CONCLUSION: In women with baseline vaginal pain, there was no difference in pain scores between the different components of the pelvic examination, nor is there a significant difference in pain during the examination compared with their baseline pain. Most patients reported minimal pain during each component.

Allergic tears promote upregulation of eosinophil adhesion to conjunctival epithelial cells in an ex vivo model: inhibition with olopatadine treatment.

PURPOSE: The mechanism by which eosinophils adhere to the ocular surface during allergic inflammation is unknown. This study examined whether the incubation of human conjunctival epithelial cells (HCEs) with tears from allergic subjects promotes eosinophil adhesion, and it examined the effect of treatment with olopatadine on this process. METHODS: Allergic subjects (n = 6) and nonallergic subjects (n = 4) were treated in season for 1 week with olopatadine in one eye while the other eye remained untreated. Tears were collected from both eyes with the use of a microcapillary tube. HCEs were acquired by enzymatic digestion of cadaveric conjunctival tissues. Confluent cultures of HCEs were treated with diluted tears for 24 hours before incubation with peripheral blood eosinophils (purified with negative magnetic bead selection). Eosinophil adhesion was measured with an eosinophil peroxidase assay. RESULTS: Incubation of HCEs with tears from allergic subjects significantly upregulated eosinophil adhesion compared with eosinophil adhesion to untreated HCEs or with HCEs treated with nonallergic tears and untreated HCEs (P < 0.05). Eosinophil adhesion to HCEs treated with tears from olopatadine-treated allergic subjects was inhibited (P < 0.01) compared with tear-stimulated adhesion observed from untreated eyes. Percentage of inhibition was 43.3% +/- 13.9% (mean +/- SD). Blocking antibodies demonstrated that eosinophil adhesion to HCEs in vitro involved beta2 integrins on eosinophils but not intercellular adhesion molecule-1 on human HCEs. CONCLUSIONS: Tears collected from allergic subjects contain bioactivity capable of upregulating eosinophil adhesion to HCEs in vitro. Inhibition of this process by treatment of subjects with olopatadine suggests that some of the cellular targets of this drug may play a role in promoting eosinophil adhesion.

Possibilities and pitfalls of outsourcing.

Outsourcing can save healthcare organizations costs related to staffing and training. Organizations should ensure that a vendor's staff is credentialed, knowledgeable, and properly trained. Outsourcing firms should ensure the confidentiality and security of the information they will handle. Outsourcing carries risks for providers, including potentially negative impact on tax-exempt status and loss of control over business processes.

Differential and cooperative effects of TNFalpha, IL-1beta, and IFNgamma on human conjunctival epithelial cell receptor expression and chemokine release.

PURPOSE: To gain better understanding of conjunctival epithelial cell responses to proinflammatory cytokines, the individual and combined effects of TNFalpha, IL-1beta, and IFNgamma on chemokine release (IL-8, regulated on activation normal T-cell expressed and secreted [RANTES]) and surface receptor expression (intercellular adhesion molecule [ICAM]-1, and HLA-DR, -DP, and -DQ) were examined. METHODS: Conjunctival epithelial cells were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent. Recombinant cytokines (0.005-50 ng/mL) were added, alone or in various combinations, 24 hours before harvesting of supernates for ELISAs and cells for flow cytometry. RESULTS: TNFalpha, IL-1beta, and IFNgamma had distinctive individual and combined effects on the parameters tested. Although TNFalpha and IL-1beta had similar and synergistic effects on increasing expression of ICAM-1, IL-1beta was a more potent upregulator of the release of IL-8 than was TNFalpha. Upregulation of IL-8 was additive when IL-1beta was combined with TNFalpha. Neither TNFalpha nor IL-1beta increased expression of HLA. In contrast, IFNgamma was a potent upregulator of both surface receptors (ICAM-1 and HLA) but IFNgamma alone had no effect on mediator release (IL-8 and RANTES). Release of RANTES required two cytokine signals, with IFNgamma and TNFalpha being the most potent combination. CONCLUSIONS: Knowledge of the differential and combined effects of proinflammatory cytokines on conjunctival epithelial cells allows better understanding of ocular inflammation.

Tear cytokines in acute and chronic ocular allergic inflammation.

PURPOSE OF REVIEW: Elevated levels of inflammatory cytokines have been reported in tears from ocular allergic disease states. The purpose of this review is to assimilate recent research contrasting tear cytokine concentrations in non-allergic subjects versus subjects with acute (seasonal allergic conjunctivitis) and chronic (giant papillary conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis) ocular allergic inflammation to discover whether the cytokine profiles could provide useful insight into disease mechanisms and therapeutic targets. RECENT FINDINGS: Recent studies have revealed distinct differences in the cytokine/chemokine concentrations in tears between the various manifestations of ocular allergy. The acute (seasonal allergic conjunctivitis) and iatrogenic (giant papillary conjunctivitis) forms of ocular allergic inflammation are characterized by an overall lack of significant cytokine changes in tears compared with chronic disease (vernal keratoconjunctivitis, atopic keratoconjunctivitis). Chronic ocular allergic inflammation produces increased concentrations of T helper 1 and 2, and proinflammatory cytokines as well as chemokines. However, vernal and atopic keratoconjunctivitis portray distinct differences in the patterns of tear cytokines/chemokines expressed. SUMMARY: The plethora of increased cytokines and chemokines in vernal and atopic keratoconjunctivitis compared with non-allergic, seasonal allergic conjunctivitis and giant papillary conjunctivitis provides a new perspective into the complex inflammatory processes occurring on the ocular surface in chronic disease. The ability to measure multiple cytokines in tears, combined with knowledge obtained from in-vitro analysis of the individual and combined effects of these cytokines on various conjunctival cells (i.e. mast cells, epithelial cells, fibroblasts) has facilitated further understanding of specific processes contributing to maintenance of inflammation and progression of vision-threatening disease and paved the way toward new therapeutic targets.

A qualitative process evaluation of classroom-based cognitive behaviour therapy to reduce adolescent depression.

Small scale trials indicate that classroom-based Cognitive Behaviour Therapy (CBT) for adolescents has good reach and can help prevent depression. However, under more diverse everyday conditions, such programmes tend not to show such positive effects. This study examined the process of implementing a classroom-based CBT depression prevention programme as part of a large (n = 5,030) randomised controlled trial across eight UK secondary schools which was not found to be effective (PROMISE, ISRCTN19083628). The views of young people (n = 42), teachers (n = 12) and facilitators (n = 16) involved in the Resourceful Adolescent Programme (RAP) were obtained via focus groups and interviews which were thematically analysed. The programme was considered to be well structured and contain useful content, particularly for younger pupils. However, challenges associated with implementation were its age appropriateness for all year groups, its perceived lack of flexibility, the consistency of quality of delivery, the competing demands for teacher time and a culture where academic targets were prioritised over personal, social and health education. Whilst schools are convenient locations for introducing such programmes and allow good reach, the culture around improving well-being of young people in schools, increasing engagement with teachers and young people and sustaining such programmes are issues that need addressing.

Teaching avian patients and caregivers in the examination room.

Client education and patient well-being should be primary goals and responsibilities for practicing avian veterinarians. Time is limited in the normal clinical appointment setting. However, this opportunity can still be used to introduce clients to the basics of training with positive reinforcement. These methods build a healthy relationship of trust between caregivers and their birds. Within the allotted appointment time, it is possible to teach clients how to train a simple behavior. This article outlines and demonstrates how training avian patients is successfully applied in a typical clinical practice.

Improving quality of health care through pay-for-performance programs.

The issue of quality of care is not new to the US health care system. Providers have been required to participate in quality improvement activities by governmental and accrediting agencies for quite some time. The public, too, is becoming increasingly involved in evaluating the quality of care provided in facilities from which they seek care. Transparency in pricing and quality of care is of critical interest to patients, health plans, and employers. On August 22, 2006, President George W. Bush signed an executive order supporting the promotion of efficient and quality health care to US citizens in health care programs administered and sponsored by the federal government, such as Medicare, Medicaid, and Tricare.However, the idea of tying reimbursement to these quality standards is growing and becoming a significant element of the health care field. Value-based purchasing refers to the many ways that health care purchasers are attempting to measure, monitor, and improve the quality of care that is received for money spent. Pay for performance is one of the emerging programs in this area. The expectation of pay for performance is that patient outcomes will be improved by rewarding providers based on predetermined measures.

Regulation of the receptor for TNFalpha, TNFR1, in human conjunctival epithelial cells.

PURPOSE: Previous studies demonstrated that mast cell-derived TNFalpha stimulation is critical to the upregulation of intercellular adhesion molecule (ICAM)-1 on human conjunctival epithelial cells (HCECs), which is an important feature of ocular allergic inflammation. Shedding of TNFR1 by TNFalpha-converting enzyme (TACE) is a primary mechanism for the regulation of TNFalpha-mediated events. This process has not been examined in HCECs. In this study, the authors examined the regulation of TNFR1 expression and shedding by TACE on primary HCECs and the IOBA-NHC conjunctival epithelial cell line. METHODS: Primary human conjunctival mast cells and epithelial cells were obtained from cadaveric conjunctival tissue. HCECs were incubated with and without activators (IgE-activated mast cell supernates, phorbol myristate acetate [PMA; to activate TACE], TNFalpha, and IFNgamma [to upregulate TNFR1]) for 24 hours. Pretreatment with the TACE inhibitor TAPI-2 was used to inhibit shedding of TNFR1. Supernates collected from the incubations were analyzed with ELISA for soluble TNFR1 (sTNFR1). With the use of flow cytometry, cells were harvested from these experiments for analysis of TNFR1 and ICAM-1 receptor expression. RESULTS: IgE-activated conjunctival mast cell supernates upregulated the expression of TNFR1. TAPI-2 inhibited the PMA-induced release of sTNFR1 receptor and enhanced the surface expression of TNFR1 in HCECs in a dose-dependent manner. Upregulation of TNFR1 expression by priming with TAPI-2 and IFNgamma resulted in enhanced ICAM-1 expression in response to TNFalpha stimulation (significant change in the slope of the dose-response curve). CONCLUSIONS: These results demonstrate that TACE promotes TNFR1 shedding in HCECs and that TNFR1 expression may be a more significant target than TNFalpha for intervention in ocular inflammation.

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses.

BACKGROUND: Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES: To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS: Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS: Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS: Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.

Pathophysiology of ocular allergy: the roles of conjunctival mast cells and epithelial cells.

Allergic eye disease is associated with IgE-mediated conjunctival inflammation leading to signs of immediate hypersensitivity, including redness, itching, and tearing. Pathologic studies using conjunctival mast cells demonstrate that these cells, when sensitized with IgE antibody and exposed to environmental allergens, release mediators involved with allergic inflammation. The type, release kinetics, and concentration of these mediators in the conjunctiva have not been completely characterized. The ability to isolate and purify mast cells and epithelial cells from human conjunctival tissue has permitted the study of mediator release and cell-to-cell signaling in this tissue. Our laboratory has developed in vitro and in vivo models to better understand how inflammatory cells are recruited to and infiltrate conjunctival tissues. These models demonstrate that mast-cell activation may supply sufficient cytokine signaling to initiate and direct the well-orchestrated trafficking of eosinophils to the ocular surface, facilitate their adhesion, and cause release of potent mediators of ocular inflammation.

The promotion of eosinophil degranulation and adhesion to conjunctival epithelial cells by IgE-activated conjunctival mast cells.

BACKGROUND: Allergen-mediated mast cell activation is a key feature of ocular allergic diseases. Evidence of eosinophil-derived mediators in tears and conjunctival biopsy specimens has been associated with chronic ocular allergic inflammation. OBJECTIVE: To examine the role of conjunctival mast cell mediators in eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. METHODS: Conjunctival cells were obtained by enzymatic digestion of cadaveric conjunctival tissues. Eosinophils were obtained from peripheral blood samples using negative magnetic bead selection. The effect of IgE-activated mast cell supernates on eosinophil degranulation and adherence to epithelial cells was compared with supernates obtained from mast cells pretreated with a degranulation inhibitor (olopatadine). Eosinophil adhesion was measured by eosinophil peroxidase assay, and eosinophil degranulation was measured by eosinophil-derived neurotoxin radioimmunoassay. RESULTS: IgE-activated conjunctival mast cell supernates stimulated adhesion of eosinophils to epithelial cells (20.4% +/- 6.3% vs 3.1% +/- 1.0%; P = .048). Degranulation was not required for this process (no effect of olopatadine). IgE-activated mast cell supernates stimulated eosinophil-derived neurotoxin release (108.89 +/- 8.27 ng/10(6) cells vs 79.45 +/- 5.21 ng/10(6) cells for controls, P = .02), which was significantly inhibited by pretreatment of mast cells with a degranulation inhibitor (79.22 +/- 4.33 ng/10(6) cells vs 61.09 +/- 5.39 ng/10(6) cells for olopatadine pretreated and untreated, respectively, P = .02). CONCLUSIONS: Mediators released from conjunctival mast cells promote eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. Degranulation inhibition studies suggest that different mast cell mediators are involved in regulation of these events.