High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

A multiplexed microfluidic system for evaluation of dynamics of immune-tumor interactions.

Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.

A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control.

The drug development pipeline is severely limited by a lack of reliable tools for prediction of human clinical safety and efficacy profiles for compounds at the pre-clinical stage. Here we present the design and implementation of a platform technology comprising multiple human cell-based tissue models in a portable and reconfigurable format that supports individual organ function and crosstalk for periods of up to several weeks. Organ perfusion and crosstalk are enabled by a precision flow control technology based on electromagnetic actuators embedded in an arrayed format on a microfluidic platform. We demonstrate two parallel circuits of connected airway and liver modules on a platform containing 62 electromagnetic microactuators, with precise and controlled flow rates as well as functional biological metrics over a two week time course. Technical advancements enabled by this platform include the use of non-sorptive construction materials, enhanced scalability, portability, flow control, and usability relative to conventional flow control modes (such as capillary action, pressure heads, or pneumatic air lines), and a reconfigurable and modular organ model format with common fluidic port architecture. We demonstrate stable biological function for multiple pairs of airway-liver models for periods of 2 weeks in the platform, with precise control over fluid levels, temperature, flow rate and oxygenation in order to support relevant use cases involving drug toxicity, efficacy testing, and organ-organ interaction.

Development of a biomimetic microfluidic oxygen transfer device.

Blood oxygenators provide crucial life support for patients suffering from respiratory failure, but their use is severely limited by the complex nature of the blood circuit and by complications including bleeding and clotting. We have fabricated and tested a multilayer microfluidic blood oxygenation prototype designed to have a lower blood prime volume and improved blood circulation relative to current hollow fiber cartridge oxygenators. Here we address processes for scaling the device toward clinically relevant oxygen transfer rates while maintaining a low prime volume of blood in the device, which is required for clinical applications in cardiopulmonary support and ultimately for chronic use. Approaches for scaling the device toward clinically relevant gas transfer rates, both by expanding the active surface area of the network of blood microchannels in a planar layer and by increasing the number of microfluidic layers stacked together in a three-dimensional device are addressed. In addition to reducing prime volume and enhancing gas transfer efficiency, the geometric properties of the microchannel networks are designed to increase device safety by providing a biomimetic and physiologically realistic flow path for the blood. Safety and hemocompatibility are also influenced by blood-surface interactions within the device. In order to further enhance device safety and hemocompatibility, we have demonstrated successful coating of the blood flow pathways with human endothelial cells, in order to confer the ability of the endothelium to inhibit coagulation and thrombus formation. Blood testing results provide confirmation of fibrin clot formation in non-endothelialized devices, while negligible clot formation was documented in cell-coated devices. Gas transfer testing demonstrates that the endothelial lining does not reduce the transfer efficiency relative to acellular devices. This process of scaling the microfluidic architecture and utilizing autologous cells to line the channels and mitigate coagulation represents a promising avenue for therapy for patients suffering from a range of acute and chronic lung diseases.

Final results of the DAPS (Denosumab Adherence Preference Satisfaction) study: a 24-month, randomized, crossover comparison with alendronate in postmenopausal women.

UNLABELLED: The final analysis of this 2-year, randomized, crossover study showed that postmenopausal women with osteoporosis were more adherent, compliant, and persistent with subcutaneous denosumab injections every 6 months than with once-weekly alendronate tablets. After receiving both treatments, women reported greater satisfaction with injectable denosumab and preferred it over oral alendronate. INTRODUCTION: Osteoporosis patients who are non-compliant or non-persistent with therapy may have suboptimal clinical outcomes. This 2-year, randomized, open-label, crossover study compared treatment adherence between subcutaneous denosumab, 60 mg every 6 months, and oral alendronate, 70 mg once weekly. METHODS: Postmenopausal women at 25 centers in the USA and Canada with bone mineral density T-scores -4.0 to -2.0 and no prior bisphosphonate use received alendronate then denosumab, or denosumab then alendronate, over successive 12-month periods. Adherence required both compliance (denosumab injections 6 months apart or ≥ 80% of alendronate tablets) and persistence (both denosumab injections or ≥ 2 alendronate doses in the last month and completion of the treatment period). RESULTS: Of the 250 women enrolled (124 alendronate, 126 denosumab), 221 entered the second year (106 denosumab, 115 alendronate). Denosumab was associated with less non-adherence than alendronate (first year, 11.9% vs 23.4%; second year, 7.5% vs 36.5%). Risk ratios for non-adherence, non-compliance, and non-persistence favored denosumab in both years (p < 0.05). Of 198 subjects expressing treatment preference, 183 (92.4%) preferred the injections over the oral therapy. BMD improved further when subjects received denosumab after alendronate and remained stable when they received alendronate after denosumab. CONCLUSION: Based on the final results of this crossover study after women had received each treatment for up to 1 year, postmenopausal women with osteoporosis were more adherent, compliant, and persistent with subcutaneous denosumab injections every 6 months than with once-weekly alendronate tablets and reported increased treatment preference and satisfaction with injectable denosumab over oral alendronate.

Adherence, preference, and satisfaction of postmenopausal women taking denosumab or alendronate.

UNLABELLED: In this study, 250 women with osteoporosis were randomized to 12 months with subcutaneous denosumab 60 mg every 6 months or oral alendronate 70 mg once weekly, then crossed over to the other treatment. The primary endpoint, treatment adherence at 12 months, was 76.6% for alendronate and 87.3% for denosumab. INTRODUCTION: The purpose of this study is to evaluate treatment adherence with subcutaneous denosumab 60 mg every 6 months or oral alendronate 70 mg once weekly. METHODS: In this multicenter, randomized, open-label, 2-year, crossover study, 250 postmenopausal women with low bone mineral density received denosumab or alendronate for 12 months, then the other treatment for 12 months. The alendronate bottle had a medication event monitoring system cap to monitor administration dates. Definitions were as follows: compliance, receiving both denosumab doses 6 (± 1) months apart or 80-100% of alendronate doses; persistence, receiving both denosumab doses and completing the month 12 visit within the visit window or ≥ 2 alendronate doses in the final month; adherence, achieving both compliance and persistence. This report includes data from the first 12 months. RESULTS: The primary study endpoint, adherence in the first 12 months, was 76.6% (95/124) for alendronate and 87.3% (110/126) for denosumab. Risk ratios for denosumab compared with alendronate at 12 months were 0.58 (p = 0.043) for non-adherence, 0.48 (p = 0.014) for non-compliance, and 0.54 (p = 0.049) for non-persistence. Subject ratings for treatment necessity, preference, and satisfaction were significantly greater for denosumab and ratings for treatment bother were significantly greater for alendronate. Adverse events were reported by 64.1% of alendronate-treated subjects and 72.0% of denosumab-treated subjects (p = 0.403). The most common adverse events were arthralgia, back pain, pain in extremity, cough, and headache (each in <10% of subjects in each group). CONCLUSIONS: Significantly greater treatment adherence was observed for subcutaneous administration of denosumab every 6 months than for oral alendronate once weekly.

Preference and satisfaction with a 6-month subcutaneous injection versus a weekly tablet for treatment of low bone mass.

UNLABELLED: The Preference and Satisfaction Questionnaire (PSQ) compares patient preference and satisfaction between a 6-month subcutaneous injection and weekly oral tablet for treatment of bone loss. Patients preferred and were more satisfied with a treatment that was administered less frequently, suggesting the acceptability of the 6-month injection for treatment of bone loss. INTRODUCTION: The PSQ compares patient preference and satisfaction between a 6-month subcutaneous injection and a weekly oral tablet for treatment of bone loss. METHODS: Postmenopausal women with low bone mass who enrolled in two separate randomized phase 3 double-blind, double-dummy studies received a 6-month subcutaneous denosumab injection (60 mg) plus a weekly oral placebo or a weekly alendronate tablet (70 mg) plus a 6-month subcutaneous placebo injection. After 12 months, patients completed the PSQ to rate their preference, satisfaction, and degree of bother with each regimen. RESULTS: Most enrolled patients (1,583 out of 1,693; 93.5%) answered >or=1 item of the PSQ. Significantly more patients preferred and were more satisfied with the 6-month injection versus the weekly tablet (P < 0.001). More patients reported no bother with the 6-month injection (90%) than the weekly tablet (62%). CONCLUSION: Patients preferred, were more satisfied, and less bothered with a 6-month injection regimen for osteoporosis.

Local drug delivery with a self-contained, programmable, microfluidic system.

The development and optimization of many new drug therapies requires long-term local delivery with controlled, but variable dosage. Current methods for chronic drug delivery have limited utility because they either cannot deliver drugs locally to a specific organ or tissue, do not permit changes in delivery rate in situ, or cannot be used in clinical trials in an untethered, wearable configuration. Here, we describe a small, self-contained system for liquid-phase drug delivery. This system enables studies lasting several months and infusion rates can be programmed and modified remotely. A commercial miniature pump is integrated with microfabricated components to generate ultralow flow rates and stroke volumes. Solutions are delivered in pulses as small as 370 nL, with pulses delivered at any interval of 1 min or longer. A unique feature of the system is the ability to infuse and immediately withdraw liquid, resulting in zero net volume transfer while compounds are exchanged by mixing and diffusion with endogenous fluid. We present in vitro results demonstrating repeatability of the delivered pulse volume for nearly 3 months. Furthermore, we present in vivo results in an otology application, infusing into the cochlea of a guinea pig a glutamate receptor antagonist, which causes localized and reversible changes in auditory sensitivity.

Concept and computational design for a bioartificial nephron-on-a-chip.

A MEMS-based, (Micro Electro Mechanical System) bioartificial device is proposed for replicating the function of a single nephron. Consistent with the anatomy and physiology of humans, our device has 3 distinct sections, replicating the function of the glomerulus, the proximal tubule, and the loop of Henle. Construction of a bioartificial loop of Henle in particular requires control of diffusion-scale features. The proposed device can be built using existing microfabrication technologies and populated with various renal cell types. A computational model is also developed to analyze the coupled, multiphase mass transport in this system. Using the model, a design is generated with flow and solute transport properties matching those of the human nephron.

Plasmablastic lymphomas may occur as post-transplant lymphoproliferative disorders.

AIMS: To describe four cases of plasmablastic lymphoma arising in the unusual setting of a post-transplantation lymphoproliferative disorder (PTLD). METHODS AND RESULTS: Four cases were encountered over 2 years in human immunodeficiency virus (HIV)-negative patients following renal, heart or bone marrow transplantation. The cases were routinely processed and immunohistochemistry was performed. The cases showed blastic non-Hodgkin's lymphoma morphology and plasma cell-like immunophenotypic features: minimal or absent expression of leucocyte common antigen and CD20, variable CD79a and VS38 positivity. Monoclonal light chain restriction was also detected. CONCLUSIONS: The emphasis of this paper is to document further the occurrence of plasmablastic lymphomas in HIV- individuals and to expand the spectrum of PTLD.

Validation of a rheumatoid arthritis health-related quality of life instrument, the CSHQ-RA.

OBJECTIVE: To test the validity and reliability of a newly developed disease-specific multidimensional quality of life instrument: the Cedars-Sinai Health-Related Quality of Life Instrument (CSHQ-RA). METHODS: A total of 350 rheumatoid arthritis (RA) patients were asked to complete the CSHQ-RA at 2 time points (4 weeks apart). Patients also completed the Medical Outcomes Study Short Form 36 (SF-36) and the Stanford Health Assessment Questionnaire (HAQ) Disability Index (DI) at the second time point. Construct validity was tested, using Pearson's correlations, by comparing subscale scores on the CSHQ-RA to those obtained from the mental component summary (MCS) and physical component summary (PCS) of the SF-36. HAQ DI scores were used to assess the discriminant validity of the CSHQ-RA. Intraclass correlation coefficients (ICCs) were used to assess test-retest reliability. RESULTS: Response rates for the first and second survey were 83% (291) and 93% (276), respectively; 84% of respondents were women, and mean age was 57 years. Mean scores +/- SDs on instruments were: HAQ 0.73 +/- 0.69; MCS 49 +/- 12; and PCS 33 +/- 11. Pearson's correlations between the CSHQ-RA subscale scores and the SF-36 scores ranged from 0.55 to 0.76 (P < 0.001). Analysis of variance indicate that scores on the CSHQ-RA discriminated between levels of physical disability as measured by the HAQ (P < 0.001). Test-retest reliability was demonstrated in the instrument's subscale scores (ICC 0.70-0.90). CONCLUSION: These results support the construct validity, discriminant validity, and reliability of the CSHQ-RA as a measure that captures the impact of RA on patients' health-related quality of life.

Identifying patients with gastroesophageal reflux disease in a managed care organization.

The ability of various strategies to identify patients with gastroesophageal reflux disease (GERD) and the relative economic impact on disease management programs for GERD were studied. A telephone interview was conducted of a random sample of patients enrolled in any of three health plans in a 100,000-member managed care organization who had either a pharmacy claim or an encounter claim during 1997. The telephone interview identified patients with GERD and served as the standard by which the sensitivity, specificity, and predictive values of the following patient-identification strategies were compared: (1) telephone interview, (2) chart review, (3) use of encounter claims, (4) use of pharmacy claims, (5) use of both encounter claims, and pharmacy claims, and (6) use of encounter claims or pharmacy claims. Conservative estimates of costs and projected savings were then used to model the potential return on investment of the strategies. A total of 1186 patients completed the telephone interview, of whom 390 (33%) met the case definition of GERD. The most sensitive method for identifying patients with GERD was using either pharmacy or encounter claims (26%). The most specific strategy with the highest positive predictive value (PPV) (87%) was using both pharmacy and encounter claims, but this approach had a case-detection rate of only 3%. Encounter claims were significantly more sensitive than pharmacy claims and yielded a higher estimate of prevalence. The telephone interview identified the most subjects who could have benefited from a disease management program and cost 84% less than chart review. While use of administrative data (pharmacy and encounter claims) was the least costly strategy, it identified 74% fewer patients expected to benefit from disease management. The efficiency of disease management programs for GERD may depend on the method of patient identification, which in turn may depend on whether PPV or negative predictive value (NPV) should be maximized. If there is a need to identify all cases (i.e., sensitivity and NPV are most important), then telephone interview may provide the greatest opportunity for disease management with the greatest return on investment, but at the expense of enrolling many patients who may not benefit.

Silicon micromachining to tissue engineer branched vascular channels for liver fabrication.

To date, many approaches to engineering new tissue have emerged and they have all relied on vascularization from the host to provide permanent engraftment and mass transfer of oxygen and nutrients. Although this approach has been useful in many tissues, it has not been as successful in thick, complex tissues, particularly those comprising the large vital organs such as the liver, kidney, and heart. In this study, we report preliminary results using micromachining technologies on silicon and Pyrex surfaces to generate complete vascular systems that may be integrated with engineered tissue before implantation. Using standard photolithography techniques, trench patterns reminiscent of branched architecture of vascular and capillary networks were etched onto silicon and Pyrex surfaces to serve as templates. Hepatocytes and endothelial cells were cultured and subsequently lifted as single-cell monolayers from these two-dimensional molds. Both cell types were viable and proliferative on these surfaces. In addition, hepatocytes maintained albumin production. The lifted monolayers were then folded into compact three-dimensional tissues. Thus, with the use microfabrication technology in tissue engineering, it now seems feasible to consider lifting endothelial cells as branched vascular networks from two-dimensional templates that may ultimately be combined with layers of parenchymal tissue, such as hepatocytes, to form three-dimensional conformations of living vascularized tissue for implantation.

The NavChair Assistive Wheelchair Navigation System.

The NavChair Assistive Wheelchair Navigation System [19] is being developed to reduce the cognitive and physical requirements of operating a power wheelchair for people with wide ranging impairments that limit their access to powered mobility. The NavChair is based on a commercial wheelchair system with the addition of a DOS-based computer system, ultrasonic sensors, and an interface module interposed between the joystick and power module of the wheelchair. The obstacle avoidance routines used by the NavChair in conjunction with the ultrasonic sensors are modifications of methods originally used in mobile robotics research. The NavChair currently employs three operating modes: general obstacle avoidance, door passage, and automatic wall following. Results from performance testing of these three operating modes demonstrate their functionality. In additional to advancing the technology of smart wheelchairs, the NavChair has application to the development and testing of "shared control" systems where a human and machine share control of a system and the machine can automatically adapt to human behaviors.

The NavBelt--a computerized travel aid for the blind based on mobile robotics technology.

This paper presents a new concept for a travel aid for the blind. A prototype device, called the NavBelt, was developed to test this concept. The device can be used as a primary or secondary aid, and consists of a portable computer, ultrasonic sensors, and stereophonic headphones. The computer applies navigation and obstacle avoidance technologies that were developed originally for mobile robots. The computer then uses a stereophonic imaging technique to process the signals from the ultrasonic sensors and relays their information to the user via stereophonic headphones. The user can interpret the information as an acoustic "picture" of the surroundings, or, depending on the operational mode, as the recommended travel direction. The acoustic signals are transmitted as discrete beeps or continuous sounds. Experimental results with the NavBelt simulator and a portable prototype show that users can travel safely in an unfamiliar and cluttered environment at speeds of up to 0.8 m/s.

Clinical practice guidelines: a tool to help provide quality care.

Significant variability of medical care exists in the United States that cannot be readily explained on the basis of severity of illness, patient demographics, or clinical necessity. One approach to promoting appropriate decision-making is to make the most recent and relevant information available to physicians in a practice guideline. Physicians are most likely to accept practice guidelines that address a specific need, are supported with scientific evidence, and offer the potential for improving patient outcomes. The implementation of a clinical practice guideline and its impact on the care of patients with uncontrolled hypertension are described.

Second-trimester membrane rupture. Abortion induced with prostaglandin E2 after oxytocin failure.

OBJECTIVE: To test an effective method of terminating second-trimester pregnancy with ruptured membranes in women who fail to abort from an oxytocin infusion. STUDY DESIGN: Five patients with rupture of membranes during the second trimester of pregnancy and failed to abort using the traditional method of intravenous oxytocin infusion were treated with intrauterine instillation of prostaglandin E2 (PGE2) solution through a double-balloon device. RESULTS: All five patients aborted within 8.8 +/- 4.5 hours from the beginning of PGE2 instillation. No major complications occurred. The only side effect was short-duration pyrexia (less than 48 hours). CONCLUSION: Use of the double-balloon device and intrauterine instillation of PGE2 was effective for termination of pregnancy in patients with rupture of membranes who do not respond to oxytocin.

Assessing individual differences in constructive versus destructive responses to anger across the lifespan.

Scenario-based, self-report measures were developed to assess how people characteristically experience and manage anger from middle childhood through adulthood. The Anger Response Inventories (ARIs) for children, adolescents, and adults each assess (a) anger arousal, (b) intentions, (c) cognitive and behavioral responses, and (d) Long-term consequences. Several independent studies provide support for the reliability and validity of the ARIs. Theoretically consistent patterns of correlations were observed with (a) global self-report measures of hostility, aggression, and anger-management strategies (adult version); (b) teacher reports of behavioral and emotional adjustment (child and adolescent versions); and (c) self- and family-member reports of behaviors on specific anger episodes (adolescent and adult versions). Findings from additional personality and developmental studies are summarized, further supporting construct validity.

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood.

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.