Retrospective analysis of pediatric patients with Burkitt lymphoma treated in Tanzania following the implementation of the 2016 National Treatment Guidelines: Poor outcomes to current standard-of-care therapy.

BACKGROUND: Despite the excellent outcomes achieved in the treatment of pediatric Burkitt lymphoma (BL) in high-income countries (HICs), outcomes remain poor in low- and middle-income countries (LMICs). Efforts to improve BL outcomes in Tanzania included the creation of National Treatment Guidelines in 2016. However, disease outcomes in Tanzania following the creation of these guidelines have not been reported to date. PROCEDURE: Historical records from 2016 to 2021 for patients 0-18 years of age with a diagnosis of BL and seen at Bugando Medical Centre (BMC), in Mwanza, Tanzania, were curated into an electronic database and analyzed descriptively. Patients in this cohort were treated per the Tanzanian National Treatment Guidelines, which include six cycles of cyclophosphamide, vincristine, and methotrexate (COM) chemotherapy with intrathecal methotrexate and cytarabine. RESULTS: In total, 92 BL patients' records were eligible for analysis. Patients in this cohort were most commonly Murphy stage II (28%) or stage III (34%). Nearly all, 91%, met International Network for Cancer Treatment and Research (INCTR) high-risk criteria at presentation. Forty-two percent of patients did not receive a biopsy and were treated with a presumed diagnosis of BL alone. A 1-year event-free survival of 29.6% (95% confidence interval [CI]: 20.3%-39.5%) and a 1-year overall survival of 38.5% (95% CI: 28%-48.9%) were observed. A high rate of treatment abandonment (34%) was also observed. CONCLUSION: In a historical cohort of pediatric patients with BL treated per the 2016 Tanzanian National Treatment Guidelines, we observed poor outcomes and a high rate of abandonment. These outcomes appear inferior to those achieved in the INCTR clinical trial that informed the guidelines' creation, and highlights the importance of "real-world" outcomes data in LMICs. These data reinforce the idea that continued clinical research and capacity building efforts are necessary to improve BL outcomes in LMICs.

Association of caregiver attitudes with adolescent HPV vaccination in 13 southern US states.

Background and objectives:  HPV vaccination coverage is lower than that of other adolescent vaccines in the southern US. This study sought to characterize caregiver attitudes associated with adolescent HPV vaccination in the southern US and to inform interventions to promote HPV vaccination. Methods: From December 2019 - January 2020, caregivers of adolescents (ages 9-17 years) living in thirteen southern US states were recruited from a nationally-representative online survey panel. Caregivers (N = 1,105) completed a cross-sectional survey that assessed general adolescent vaccine attitudes as well as those associated with the HPV vaccine and HPV vaccination decision-making. The primary study outcome was adolescents' receipt of at least one dose of the HPV vaccine. Results: Caregivers with vaccinated adolescents had greater positive attitudes towards adolescent vaccines compared to caregivers of unvaccinated adolescents. Top three areas of concern among caregivers were related to vaccine ingredients, perceptions that adolescents receive too many vaccines, and worry about vaccine side effects. In multivariable regression models, positive attitudes towards the HPV vaccine and HPV vaccination decision-making strongly associated with HPV vaccination in addition to general adolescent vaccination attitudes. Caregivers' reported discomfort with discussing the topic of sex was predictive of lower vaccination uptake for older adolescents. Conclusions: Public health messaging in the southern US should be tailored to reduce concerns about vaccine safety and to communicate the importance of timely HPV vaccination. Campaigns that deliver information specific to the HPV vaccine and to support vaccination decision-making may be more effective than those delivering only general adolescent vaccination information at promoting on-time HPV vaccination.

Using the Consolidated Framework for Implementation Research to Inform the Design of the Mobile Inspección Visual con Ácido Acético System: Mixed Methods Case Study.

BACKGROUND: There is growing evidence supporting the use of mobile health (mHealth) interventions in low- and middle-income countries to address resource limitations in the delivery of health information and services to vulnerable populations. In parallel, there is an increasing emphasis on the use of implementation science tools and frameworks for the early identification of implementation barriers and to improve the acceptability, appropriateness, and adoption of mHealth interventions in resource-limited settings. However, there are limited examples of the application of implementation science tools and frameworks to the formative phase of mHealth design for resource-limited settings despite the potential benefits of this work for enhancing subsequent implementation, scale-up, and sustainability. OBJECTIVE: We presented a case study on the use of an implementation science framework in mHealth design. In particular, we illustrated the usability of the Consolidated Framework for Implementation Research (CFIR) for organizing and interpreting formative research findings during the design of the mobile Inspección Visual con Ácido Acético (mIVAA) system in Lima, Peru. METHODS: We collected formative data from prospective users of the mIVAA intervention using multiple research methodologies, including structured observations, surveys, group and individual interviews, and discussions with local stakeholders at the partnering organization in Peru. These activities enabled the documentation of clinical workflows, perceived barriers to and facilitators of mIVAA, overarching barriers to cervical cancer screening in community-based settings, and related local policies and guidelines in health care. Using a convergent mixed methods analytic approach and the CFIR as an organizing framework, we mapped formative research findings to identify key implementation barriers and inform iterations of the mIVAA system design. RESULTS: In the setting of our case study, most implementation barriers were identified in the CFIR domains of intervention characteristics and inner setting. All but one barrier were addressed before mIVAA deployment by modifying the system design and adding supportive resources. Solutions involved improvements to infrastructure, including cellular data plans to avoid disruption from internet failure; improved process and flow, including an updated software interface; and better user role definition for image capture to be consistent with local health care laws. CONCLUSIONS: The CFIR can serve as a comprehensive framework for organizing formative research data and identifying key implementation barriers during mHealth intervention design. In our case study of the mIVAA system in Peru, formative research contributing to the CFIR domains of intervention characteristics and inner setting elicited the most key barriers to implementation. The early identification of barriers enabled design iterations before system deployment. Future efforts to develop mHealth interventions for low- and middle-income countries may benefit from using the approach presented in this case study as well as prioritizing the CFIR domains of intervention characteristics and inner setting.

Key stakeholder perspectives on challenges and opportunities for rural HPV vaccination in North and South Carolina.

The objective of this study was to identify factors at the individual, provider, and systems levels that serve as challenges or opportunities for increasing adolescent vaccination-including Human Papillomavirus (HPV) vaccination-in rural communities in the southern United States (US). As part of a broader study to increase HPV vaccine uptake in the southern US, we conducted in-depth interviews with vaccination stakeholders representing public health and education agencies in North Carolina (NC) and South Carolina (SC). Fourteen key stakeholders were recruited using purposive sampling to obtain insights into challenges and solutions to rural-urban disparities in HPV vaccination coverage. Stakeholders were also queried about their experiences and attitudes toward school-based vaccination promotion programs and campaigns. We used a rapid qualitative approach to analyze the data. Stakeholders identified factors at the individual, provider, and systems levels that serve as challenges to vaccination in rural communities. Similar to previous studies, stakeholders mentioned challenges with healthcare access and vaccine-related misconceptions that pose barriers to HPV vaccination for rural residents. Systems-level challenges identified included limited access to high-speed internet in rural areas that may impact providers' ability to interface with state-level digital systems such as the vaccination registry. Stakeholders identified a number of opportunities to increase HPV vaccination coverage, including through school-based health promotion programs. Stakeholders strongly supported school-based programs and approaches to strengthen confidence and demand for HPV vaccination and to help address persistent social determinants and system level factors that pose challenges to HPV vaccination coverage in many rural areas.

Caregiver Acceptability of Mobile Phone Use for Pediatric Cancer Care in Tanzania: Cross-sectional Questionnaire Study.

BACKGROUND: There is a 60% survival gap between children diagnosed with cancer in low- and middle-income countries (LMICs) and those in high-income countries. Low caregiver knowledge about childhood cancer and its treatment results in presentation delays and subsequent treatment abandonment in LMICs. However, in-person education to improve caregiver knowledge can be challenging due to health worker shortages and inadequate training. Due to the rapid expansion of mobile phone use worldwide, mobile health (mHealth) technologies offer an alternative to delivering in-person education. OBJECTIVE: The aim of this study is to assess patterns of mobile phone ownership and use among Tanzanian caregivers of children diagnosed with cancer as well as their acceptability of an mHealth intervention for cancer education, patient communication, and care coordination. METHODS: In July 2017, caregivers of children <18 years diagnosed with cancer and receiving treatment at Bugando Medical Centre (BMC) were surveyed to determine mobile phone ownership, use patterns, technology literacy, and acceptability of mobile phone use for cancer education, patient communication, and care coordination. Descriptive statistics were generated from the survey data by using mean and SD values for continuous variables and percentages for binary or categorical variables. RESULTS: All eligible caregivers consented to participate and completed the survey. Of the 40 caregivers who enrolled in the study, most used a mobile phone (n=34, 85%) and expressed high acceptability in using these devices to communicate with a health care provider regarding treatment support (n=39, 98%), receiving laboratory results (n=37, 93%), receiving reminders for upcoming appointments (n=38, 95%), and receiving educational information on cancer (n=35, 88%). Although only 9% (3/34) of mobile phone owners owned phones with smartphone capabilities, about 74% (25/34) self-reported they could view and read SMS text messages. CONCLUSIONS: To our knowledge, this is the first study to assess patterns of mobile phone ownership and use among caregivers of children with cancer in Tanzania. The high rate of mobile phone ownership and caregiver acceptability for a mobile phone-based education and communication strategy suggests that a mobile phone-based intervention, particularly one that utilizes SMS technology, could be feasible in this setting.

Disparities in healthcare access and utilization and human papillomavirus (HPV) vaccine initiation in the United States.

Currently in the United States, Human Papillomavirus (HPV) vaccination coverage among eligible individuals is lower compared to coverage goals of 80% set by the HealthyPeople 2030 initiative. In this study, we used the National Health and Nutrition Examination Survey (NHANES) 2015-2016 and 2017-2018 datasets to determine the association between HPV vaccine initiation among individuals of ages 9 to 26 years and their patterns of healthcare access and utilization. In particular, we examined the following healthcare characteristics: 1) having a routine place of healthcare, 2) having health insurance coverage, 3) frequency of healthcare visits per year, and 4) type of routine place of healthcare (outpatient primary care vs. ED, etc.). We fit independent multivariable logistic regression models for each NHANES dataset and controlled for sociodemographic characteristics and interactions with healthcare access and utilization characteristics. Our findings suggest that HPV vaccine initiation is positively associated with having a routine place of healthcare (2015-2016: aOR 1.92, 95% CI 1.25-2.95; 2017-2018: aOR 1.99, 95% CI 1.07-3.68). Relatedly, HPV vaccine initiation is negatively associated with never having received healthcare in the past year (2015-2016: aOR 0.61, 95% CI 0.41-0.90; 2017-2018: aOR 0.45, 95% CI 0.27-0.75). The results of this study suggest that interventions to promote HPV vaccination should include strategies that promote access to and utilization of routine health care services. Our findings are particularly salient in light of the drop in HPV vaccine initiation and healthcare access and utilization among adolescents during the COVID-19 pandemic.

Predictors of HPV vaccination in the southern US: A survey of caregivers from 13 states.

BACKGROUND AND OBJECTIVES: Despite a high burden of human papillomavirus (HPV)-attributable cancers, the southern US lags other regions in HPV vaccination coverage. This study sought to characterize and contextualize predictors of HPV vaccination in the southern US. METHODS: From December 2019 - January 2020, parents of adolescents (ages 9-17 years) living in thirteen southern US states were recruited from a nationally-representative online survey panel and completed a cross-sectional survey. The primary study outcome was initiation of HPV vaccination. RESULTS: Of 1105 parents who responded to the survey, most were ≥35 years of age and of female gender. HPV vaccination initiation was reported only among 37.3% of adolescents and was highest at age 12. Cumulative HPV vaccination coverage was highest at age 15 (60%) but lower than coverage for tetanus-diphtheria-acellular pertussis (Tdap, 79.3%) and Meningococcal vaccines (MenACWY, 67.3%). Provider recommendation was strongly associated with higher odds of HPV vaccination (aOR: 49.9, 95 %CI: 23.1-107.5). In alternative predictive models, home/online (vs. public) schooling and parents' working status were associated with lower odds of vaccination; health care visits in the past 12 months and shorter travel times to adolescents' usual health care provider were associated with greater odds of vaccination. CONCLUSIONS: Our findings suggest missed opportunities for HPV vaccination in the southern US and support strengthening provider recommendation for on-time initiation of HPV vaccination among adolescents. Other strategies to increase HPV vaccinations may include encouraging co-administration with other adolescent vaccines, increasing vaccine access, and promoting vaccinations for home/online-school students.

Mucosa-adherent Pediococcus Pentosaceus I44 isolated from healthy human and effect of oleic acid on its probiotic properties.

With the aim of selectively isolating and understanding the cultivable lactic acid bacteria that are autochthonous to human gut, biopsy samples were collected from the terminal ileum and caecum of healthy volunteers who underwent routine colonoscopy procedure. The use of tissue samples (over stool samples) provides for a better expression of the autochthonous bacterial population of the human gut. The strains that were stable after many rounds of sub-culture were identified and studied further. One such ileal isolate, a Gram-positive, catalase-negative cocci was identified to be Pediococcus pentosaceus I44. It was studied for its gastric tolerance, bile tolerance and surface properties, and reported here. The effect of tween-80, oleic acid, extra virgin olive oil and sesame oil on the aforementioned properties of P. pentosaceus I44 was also analysed. The isolate was able to survive well in simulated gastric fluid of pH 3.0 followed by treatment in simulated intestinal fluid with a survival percentage of around 70%. It was, however, unable to withstand pH 2.0 even in the presence of supplements. It showed good tolerance to bile stress (1% w/v), and its cell surface was found to be hydrophilic. P. pentosaceus I44 showed good aggregation of 87% after 24 h, with oleic acid having a significant effect on the isolate's aggregation potential. P. pentosaceus I44 is one of the few mucosa-bound cultivable bacteria that are adapted for survival in human colon. Studying and analysing such isolates might provide useful insights into their role in metabolism and health.

Examining Associations between Knowledge and Vaccine Uptake Using the Human Papillomavirus Knowledge Questionnaire (HPV-KQ).

Objectives: Understanding the relationship between human papillomavirus (HPV) knowledge and vaccination behavior is important to inform public health interventions, yet few validated HPV knowledge scales exist. This study describes development of the Human Papillomavirus Knowledge Questionnaire (HPV-KQ) and its validation with parents residing in the southern United States (US). Methods: Drawing on previously published measures, we developed the 13-item HPV-KQ and administered the scale via Web-based survey to parents (N=1105) of adolescents ages 9 to 17 years. Dimensionality, internal consistency, model fit, and predictive validity were assessed. Results: The scale was bidimensional. One factor captured general HPV knowledge, and the second factor captured perceptions of gender differences in HPV infection and vaccine recommendations. The 13-item scale and 2-factor solution displayed strong internal consistency and good model fit. Parents of vaccinated adolescents scored higher on the 13-item HPV-KQ (Mean = 8.56) than parents of unvaccinated adolescents (Mean = 6.43) (p < .001). In regression models, controlling for key covariates, parents' performance on the HPV-KQ predicted adolescent HPV vaccination (p < .001). Conclusions: Evaluation indicates the HPV-KQ is a reliable and valid tool for measuring knowledge of HPV and the HPV vaccine among parents residing in the southern US. We recommend further efforts to validate the scale with other populations.

Knowledge, attitudes, and practices regarding cervical cancer screening among women in metropolitan Lima, Peru: a cross-sectional study.

BACKGROUND: Cervical cancer is a leading cause of cancer deaths among women of reproductive age in Peru. Screening and early identification of pre-cancerous lesions are a cornerstone of the cervical cancer prevention strategy. Yet, there is limited literature on barriers to screening among Peruvian women. In this cross-sectional study, we aimed to examine Peruvian women's knowledge, attitudes, and practices regarding cervical cancer screening and identify possible reasons for the gap between knowledge and screening. METHODS: The study was conducted in metropolitan Lima from June-August 2019. We purposefully recruited 12 women who had previously been screened, and 12 who had never been screened for cervical cancer. The women completed a 40-question knowledge and attitude survey and an in-depth interview about barriers to screening. Descriptive analysis was used to calculate a knowledge and attitude score and qualitative analysis was guided by the Health Belief Model constructs. RESULTS: Previously screened participants had greater knowledge of cervical cancer symptoms, risk factors, and prevention (mean score = 28.08, S.D. = 4.18) compared to participants who had never been screened (mean score = 21.25, S.D. = 6.35). Both groups described lack of priority and embarrassment as barriers to cervical cancer screening. For participants who had never been screened before, major barriers included the fear of a cancer diagnosis and lack of information about screening services. Pregnancy, unusual gynecological symptoms and encouragement from friends and family were cues to action for participants seeking screening. Most participants in both groups recognized the benefits of getting screened for cervical cancer. Being previously screened increased participants' self-efficacy for engaging in screening behaviors again. Misconceptions regarding screening procedures and cervical cancer were also noted as barriers for participants accessing screening services. CONCLUSIONS: Improving knowledge and awareness about cervical cancer and screening programs may improve screening behaviors among women. Targeting women who have never been screened before and addressing their fears and concerns around embarrassment may be other areas for intervention. Misconceptions that deter women from screening services are an important issue that should be addressed in order to increase the number of women who get timely screenings.

Digital Health Strategies for Cervical Cancer Control in Low- and Middle-Income Countries: Systematic Review of Current Implementations and Gaps in Research.

BACKGROUND: Nearly 90% of deaths due to cervical cancer occur in low- and middle-income countries (LMICs). In recent years, many digital health strategies have been implemented in LMICs to ameliorate patient-, provider-, and health system-level challenges in cervical cancer control. However, there are limited efforts to systematically review the effectiveness and current landscape of digital health strategies for cervical cancer control in LMICs. OBJECTIVE: We aim to conduct a systematic review of digital health strategies for cervical cancer control in LMICs to assess their effectiveness, describe the range of strategies used, and summarize challenges in their implementation. METHODS: A systematic search was conducted to identify publications describing digital health strategies for cervical cancer control in LMICs from 5 academic databases and Google Scholar. The review excluded digital strategies associated with improving vaccination coverage against human papillomavirus. Titles and abstracts were screened, and full texts were reviewed for eligibility. A structured data extraction template was used to summarize the information from the included studies. The risk of bias and data reporting guidelines for mobile health were assessed for each study. A meta-analysis of effectiveness was planned along with a narrative review of digital health strategies, implementation challenges, and opportunities for future research. RESULTS: In the 27 included studies, interventions for cervical cancer control focused on secondary prevention (ie, screening and treatment of precancerous lesions) and digital health strategies to facilitate patient education, digital cervicography, health worker training, and data quality. Most of the included studies were conducted in sub-Saharan Africa, with fewer studies in other LMIC settings in Asia or South America. A low risk of bias was found in 2 studies, and a moderate risk of bias was found in 4 studies, while the remaining 21 studies had a high risk of bias. A meta-analysis of effectiveness was not conducted because of insufficient studies with robust study designs and matched outcomes or interventions. CONCLUSIONS: Current evidence on the effectiveness of digital health strategies for cervical cancer control is limited and, in most cases, is associated with a high risk of bias. Further studies are recommended to expand the investigation of digital health strategies for cervical cancer using robust study designs, explore other LMIC settings with a high burden of cervical cancer (eg, South America), and test a greater diversity of digital strategies.

Barriers to the uptake of cervical cancer services and attitudes towards adopting new interventions in Peru.

Cervical cancer mortality is high among Peruvian women of reproductive age. Understanding barriers and facilitators of cervical cancer screening and treatment could facilitate development of contextually-relevant interventions to reduce cervical cancer incidence and mortality. From April to October 2019, we conducted a cross-sectional survey with 22 medical personnel and administrative staff from Liga Contra el Cancer, in Lima, Peru. The survey included structured and open-ended questions about participants' roles in cervical cancer prevention and treatment, perceptions of women's barriers and facilitators for getting screened and/or treated for cervical cancer, as well as attitudes towards adopting new cervical cancer interventions. For structured questions, the frequency of responses for each question was calculated. For responses to open-ended questions, content analysis was used to summarize common themes. Our data suggest that the relative importance and nature of barriers that Peruvian women face are different for cervical cancer screening compared to treatment. In particular, participants mentioned financial concerns as the primary barrier to treatment and a lack of knowledge or awareness of human papillomavirus and/or cervical cancer as the primary barrier to screening uptake among women. Participants reported high willingness to adopt new interventions or strategies related to cervical cancer. Building greater awareness about benefits of cervical cancer screening among women, and reducing financial and geographic barriers to treatment may help improve screening rates, decrease late-stage diagnosis and reduce mortality in women who have a pre-cancer diagnosis, respectively. Further studies are needed to generalize study findings to settings other than Lima, Peru.

Using digital health to facilitate compliance with standardized pediatric cancer treatment guidelines in Tanzania: protocol for an early-stage effectiveness-implementation hybrid study.

BACKGROUND: In high-income countries (HICs), increased rates of survival among pediatric cancer patients are achieved through the use of protocol-driven treatment. Compared to HICs, differences in infrastructure, supportive care, and human resources, make compliance with protocol-driven treatment challenging in low- and middle-income countries (LMICs). For successful implementation of protocol-driven treatment, treatment protocols must be resource-adapted for the LMIC context, and additional supportive tools must be developed to promote protocol compliance. In Tanzania, an LMIC where resource-adapted treatment protocols are available, digital health applications could promote protocol compliance through incorporation of systematic decision support algorithms, reminders and alerts related to patient visits, and up-to-date data for care coordination. However, evidence on the use of digital health applications in improving compliance with protocol-driven treatment for pediatric cancer is limited. This study protocol describes the development and evaluation of a digital health application, called mNavigator, to facilitate compliance with protocol-driven treatment for pediatric cancer in Tanzania. METHODS: mNavigator is a digital case management system that incorporates nationally-approved and resource-adapted treatment protocols for two pediatric cancers in Tanzania, Burkitt lymphoma and retinoblastoma. mNavigator is developed from an open-source digital health platform, called CommCare, and guided by the Consolidated Framework for Implementation Research. From July 2019-July 2020 at Bugando Medical Centre in Mwanza, Tanzania, all new pediatric cancer patients will be registered and managed using mNavigator as the new standard of care for patient intake and outcome assessment. Pediatric cancer patients with a clinical diagnosis of Burkitt lymphoma or retinoblastoma will be approached for participation in the study evaluating mNavigator. mNavigator users will document pre-treatment and treatment details for study participants using digital forms and checklists that facilitate compliance with protocol-driven treatment. Compliance with treatment protocols using mNavigator will be compared to historical compliance rates as the primary outcome. Throughout the implementation period, we will document factors that facilitate or inhibit mNavigator implementation. DISCUSSION: Study findings will inform implementation and scale up of mNavigator in tertiary pediatric cancer facilities in Tanzania, with the goal of facilitating protocol-driven treatment. TRIAL REGISTRATION: The study protocol was registered in ClinicalTrials.gov (NCT03677128) on September 19, 2018.

The beta- and gamma-isoforms of type I PIP5K regulate distinct stages of Ca2+ signaling in mast cells.

Crosslinking of IgE receptors by antigen initiates Ca2+ mobilization in mast cells by activating phospholipase-C gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. The resulting inositol 1,4,5-trisphosphate-mediated Ca2+ release from the endoplasmic reticulum (ER) activates store-operated Ca2+ entry, which is necessary for exocytotic release of inflammatory mediators. To investigate roles for PtdIns(4,5)P2-synthesizing isozymes of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIP5K-I) in mast cell signaling, we compared the ectopic expression of wild-type and catalytically inactive PIP5K-I beta in RBL-2H3 mast cells. Surprisingly, both antigen and thapsigargin-stimulated Ca2+ influx were reduced by overexpression of active PIP5K-I beta, whereas antigen-stimulated Ca2+ release from ER stores was unaffected. Consistent with these results, Ca2+ entry stimulated by antigen or thapsigargin was enhanced by expression of a plasma-membrane-associated inositol polyphosphate 5'-phosphatase, whereas antigen-stimulated Ca2+ release from stores was reduced. To investigate the role of PIP5K-I gamma in antigen-stimulated Ca2+ mobilization, we used bone-marrow-derived mast cells from PIP5K-I gamma(-/-) mice. Antigen-stimulated Ca2+ release from ER stores was substantially reduced in the absence of PIP5K-I gamma, but thapsigargin-mediated Ca2+ entry was unaffected. In summary, PIP5K-I gamma positively regulates antigen-stimulated Ca2+ release from ER stores, whereas PIP5K-I beta negatively regulates store-operated Ca2+ entry, suggesting that these different PIP5K-I isoforms synthesize functionally distinct pools of PtdIns(4,5)P2 at the plasma membrane.

Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays.

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.