OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

Myeloperoxidase Gene Deletion Causes Drastic Microbiome Shifts in Mice and Does Not Mitigate Dextran Sodium Sulphate-Induced Colitis.

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

Epithelial dendritic cells vs. Langerhans cells: Implications for mucosal vaccines.

Next-generation vaccines may be delivered via the skin and mucosa. The stratified squamous epithelium (SSE) represents the outermost layer of the skin (epidermis) and type II mucosa (epithelium). Langerhans cells (LCs) have been considered the sole antigen-presenting cells (APCs) to inhabit the SSE; however, it is now clear that dendritic cells (DCs) are also present. Importantly, there are functional differences in how LCs and DCs take up and process pathogens as well as their ability to activate and polarize T cells, though whether DCs participate in neuroimmune interactions like LCs is yet to be elucidated. A correct definition and functional characterization of APCs in the skin and anogenital tissues are of utmost importance for the design of better vaccines and blocking pathogen transmission. Here, we provide a historical perspective on the evolution of our understanding of the APCs that inhabit the SSE, including a detailed review of the most recent literature.

OMIP-096: A 24-color flow cytometry panel to identify and characterize CD4+ and CD8+ tissue-resident T cells in human skin, intestinal, and type II mucosal tissue.

There is a great need to understand human immune cells within tissue, where disease manifests and infection occurs. Tissue-resident memory T cells (TRMs) were discovered over a decade ago, there is a great need to understand their role in human disease. We developed a 24-color flow cytometry panel to comprehensively interrogate CD4+ and CD8+ TRMs isolated from human tissues. When interrogating cells within human tissue, enzymatic methods used to liberate cells from within the tissue can cause cleavage of cell surface markers needed to phenotype these cells. Here we carefully select antibody clones and evaluate the effect of enzymatic digestion on the expression of markers relevant to the identification of T cell residency, as well as markers relevant to the activation and immunoregulation status of these cells. We have designed this panel to be applicable across a range of human tissues including skin, intestine, and type II mucosae such as the vagina.

Factors Associated With Benzodiazepine and Antipsychotic Prescribing in Hospice: A Qualitative Study of Hospice Prescribers.

OBJECTIVES: Benzodiazepine and antipsychotic medications are common components of the hospice toolkit and are routinely prescribed for behavioral symptom management at end of life. These medications have significant associated risks but, despite their frequent use, little is known about how clinicians weigh prescribing decisions for individuals in hospice. In this qualitative study, we examined the key factors that influence the decision to initiate a benzodiazepine and antipsychotic medication for management of behavioral symptoms at end of life. DESIGN: A qualitative study using semi-structured interviews and descriptive qualitative analysis. SETTING AND PARTICIPANTS: We conducted semi-structured interviews with prescribing hospice physicians and nurse practitioners working in hospice settings across the United States. METHODS: Hospice clinicians were asked to describe factors that influence prescribing decisions to initiate benzodiazepine and antipsychotic medications for the management of behavioral symptoms. Data from audio-recorded sessions were transcribed, coded to identify relevant concepts, and reduced to determine major themes. RESULTS: We completed 23 interviews with hospice physicians and nurse practitioners. On average, participants had worked in a hospice setting for a mean of 14.3 years (SD: 10.9); 39% had geriatrics training. Major themes related to benzodiazepine and antipsychotic prescribing were (1) caregiving factors strongly influence the use of medications, (2) patient and caregiver stigma and concerns regarding medication use limit prescribing, (3) medications are initiated to avoid hospitalization or transition to a higher level of care, and (4) nursing home hospice care brings unique challenges. CONCLUSION AND IMPLICATIONS: Caregiver factors and the setting of hospice care strongly influence clinician decisions to initiate benzodiazepines and antipsychotics in hospice. Caregiver education about medication use at end of life and support in managing challenging behaviors may help promote optimal prescribing.

Association of Neoadjuvant Pembrolizumab for Oral Cavity Squamous Cell Carcinoma With Adverse Events After Surgery in Treatment-Naive Patients.

Importance: Pembrolizumab, a monoclonal antibody targeting programmed cell death 1, is currently approved by the US Food and Drug Administration for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). The potential neoadjuvant role of programmed cell death 1 inhibitors in primary surgical management of HNSCC and effects on surgical outcomes are poorly understood. Objective: To evaluate the incidence of postoperative adverse events in treatment-naive patients with advanced oral cavity cancer receiving neoadjuvant pembrolizumab when compared with matched controls, as part of a window-of-opportunity multi-institutional clinical trial assessing neoadjuvant pembrolizumab for locally advanced HNSCC. Design, Setting, and Participants: This retrospective cohort study at a single tertiary academic institution included treatment-naive patients with local regionally advanced oral cavity squamous cell carcinoma (OCSCC) who were undergoing surgical resection. Exposures: Patients with local regionally advanced resectable OCSCC who received neoadjuvant pembrolizumab were retrospectively reviewed for postoperative adverse events. Controls were matched by age, race, smoking status, and overall cancer stage based on historical data at the same institution. Matched-cohort analysis was performed using a McNemar test to assess differences between the groups. Main Outcomes and Measures: Incidence of adverse events following surgical resection of advanced OCSCC within 30 days of surgery and on continued follow-up. Results: A total of 64 patients (32 as part of the prospective clinical trial and 32 as controls; mean [SD] age, 59.6 [10.3] years; 28 [44%] women) were included in the analysis. Postoperative adverse events in the 32 patients receiving pembrolizumab included lymphedema (n = 20 [63%]), trismus (n = 7 [22%]), return to operating room (n = 7 [22%]), wound infection (n = 7 [22%]), fistula (n = 6 [19%]), wound dehiscence (n = 4 [13%]), flap failure (n = 3 [9%]), and hematoma (n = 2 [6%]). The matched control group demonstrated similar complication rates without considerable differences, except for trismus (n = 16 [50%]), which was greater by a difference of 28.1% (95% CI, 5.6%-50.6%) in the control group. Conclusions and Relevance: This cohort study examined surgical complications among patients with local regionally advanced OCSCC treated with neoadjuvant pembrolizumab and found that serious adverse events were similar to those in patients who underwent standard-of-care treatment. This suggests that there is no increased perioperative morbidity in the use of preoperative treatment with immunotherapy. Further prospective studies are needed to validate these findings for oral cavity cancer and other subsites of the head and neck.

Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

The Role of Tissue Resident Memory CD4 T Cells in Herpes Simplex Viral and HIV Infection.

Tissue-resident memory T cells (TRM) were first described in 2009. While initially the major focus was on CD8+ TRM, there has recently been increased interest in defining the phenotype and the role of CD4+ TRM in diseases. Circulating CD4+ T cells seed CD4+ TRM, but there also appears to be an equilibrium between CD4+ TRM and blood CD4+ T cells. CD4+ TRM are more mobile than CD8+ TRM, usually localized deeper within the dermis/lamina propria and yet may exhibit synergy with CD8+ TRM in disease control. This has been demonstrated in herpes simplex infections in mice. In human recurrent herpes infections, both CD4+ and CD8+ TRM persisting between lesions may control asymptomatic shedding through interferon-gamma secretion, although this has been more clearly shown for CD8+ T cells. The exact role of the CD4+/CD8+ TRM axis in the trigeminal ganglia and/or cornea in controlling recurrent herpetic keratitis is unknown. In HIV, CD4+ TRM have now been shown to be a major target for productive and latent infection in the cervix. In HSV and HIV co-infections, CD4+ TRM persisting in the dermis support HIV replication. Further understanding of the role of CD4+ TRM and their induction by vaccines may help control sexual transmission by both viruses.

Management of Wound Myiasis in the Hospice and Palliative Medicine Setting.

Complex wounds are common complications in hospice and palliative medicine (HPM), especially in patients with aggressive malignancies. Myiasis, or an infestation of maggots, is a rare but significant complication of such wounds. While uncommon in the United States, many HPM patients have multiple risk factors and comorbidities that increase their vulnerability to this condition. Currently, there are no standard diagnostic or treatment guidelines for wound myiasis. In addition, common management strategies may not be easily accessible in HPM settings. We present this case of a patient with malignant squamous cell carcinoma of the neck complicated by myiasis while in hospice, and our experience diagnosing and managing her infestation. We also reflect on special considerations for HPM patients when addressing the physical and psychological symptoms of wound myiasis.

The Role of Self-Care in Clinical Ethics Consultation: Clinical Ethicists' Risk for Burnout, Potential Harms, and What Ethicists Can Do.

Clinical ethics consultants are inevitably called to participate in and bear witness to emotionally challenging cases. With the move toward the professionalization of ethics consultants, the responsibility to respond to and address difficult ethical dilemmas is likely to fall to a small set of people or a single clinical ethicist. Combined with time constraints, the urgent nature of these cases, and the moral distress of clinicians and staff encountered during consultation, like other healthcare professionals such as physicians and nurses, clinical ethics consultants could risk burnout. If it is true that clinical ethicists are at risk for burnout, an important strategy to avoid burnout is to develop sound self-care practices. This article reviews the goals and skills of ethics consultation and the role-specific reasons that clinical ethicists may be at risk for burnout, and argues that clinical ethicists may need to engage in self-care practices. Strategies to address burnout are reviewed and opportunities for future research are identified.

Transparent Films Made of Highly Scattering Particles.

Today, colloids are widely employed in various products from creams and coatings to electronics. The ability to control their chemical, optical, or electronic features by controlling their size and shape explains why these materials are so widely preferred. Nevertheless, altering some of these properties may also lead to some undesired side effects, one of which is an increase in optical scattering upon concentration. Here, we address this strong scattering issue in films made of binary colloidal suspensions. In particular, we focus on raspberry-type polymeric particles made of a spherical polystyrene core decorated by small hemispherical domains of acrylate with an overall positive charge, which display an unusual stability against aggregation in aqueous solutions. Their solid films display a brilliant red color due to Bragg scattering but appear completely white on account of strong scattering otherwise. To suppress the scattering and induce transparency, we prepared films by hybridizing them with oppositely charged PS particles with a size similar to that of the bumps on the raspberries. We report that the smaller PS particles prevent raspberry particle aggregation in solid films and suppress scattering by decreasing the spatial variation of the refractive index inside the film. We believe that the results presented here provide a simple strategy to suppress strong scattering of larger particles to be used in optical coatings.

Differences in Adverse Effect Profiles of Corticosteroids in Palliative Care Patients.

BACKGROUND:: Corticosteroids are frequently utilized in the palliative care setting to combat symptoms such as fatigue, dyspnea, pain, weakness, anorexia, cachexia, nausea, and vomiting. Often times, adverse effects arise with corticosteroid use, and it is unclear whether switching to another corticosteroid would reduce the risk of specific adverse effects or what measures can be taken to alleviate the adverse effects. OBJECTIVE:: This article aims to review the differentiating pharmacokinetics, potency, and adverse effect profiles of corticosteroids and summarize their clinical applicability. METHODS:: A literature review of "corticosteroids" and "palliative care" was performed using the PubMed database through July 2018. Original studies relevant to the purpose of this study were identified and those that met inclusion criteria were included. RESULTS:: Although corticosteroids share many common factors, including similar pharmacokinetic, pharmacodymanic, and adverse effect profiles, they have significant differences when the details of these variables are reviewed. Providers that prescribe corticosteroids for symptom management should be aware of these differences and the recommended management strategies. CONCLUSIONS:: Recognition of corticosteroid induced adverse effect profiles and possible management strategies is crucial to optimal symptom management in palliative care patients.

Electing Full Code in Hospice: Patient Characteristics and Live Discharge Rates.

BACKGROUND: It is unknown how many hospice enrollees elect to be full code and if this is associated with higher hospice live discharge rates. OBJECTIVE: To measure the rates of hospice enrollees electing full code, the characteristics predicting full code status, and the association of full code status with various hospice live discharge patterns. DESIGN: Retrospective cohort study of electronic medical record data. SETTING/SUBJECTS: A total of 25,636 decedents enrolled in two Michigan hospices between 2009 and 2014. MEASUREMENTS: Code status was defined as full code versus do-not-resuscitate (DNR) orders. Covariates include demographics, location (home, hospice facility, nursing home, and hospital), primary diagnosis, and length of stay. Hospice live discharge was defined as short (0-14 days), medium (15-179 days), and long (>179 days). RESULTS: A total of 12.9% of hospice enrollees elected full code status. This was significantly (p < 0.05) predicted by male sex, younger age, nonwhite race, home setting of care, and cancer diagnosis. Those with full code status had 1.76 times the adjusted odds of hospice live discharge compared with those with DNR orders (95% confidence interval [CI] 1.44-2.16) and 2.47 times the odds of short live discharge (95% CI 1.69-3.62) with no significant difference in long live discharge. The association of full code orders with hospice live discharge was stronger for nonwhite enrollees, with a live discharge rate of 23.8% versus 11.6% for African Americans with full code versus DNR orders. CONCLUSIONS: Those electing full code status on admission to hospice are at high risk of live hospice discharge after short enrollments, particularly nonwhite enrollees.

The Value of a Brief Thought for the Day Reflection on an Academic Consult Service.

BACKGROUND: Work in hospice and palliative medicine can be stressful. A variety of methods have been used to mitigate workplace stress including mindfulness mediation, reflective writing, and physical activity. An intervention implemented at our institution is a "Thought for the Day," a short reflection on a piece of poetry, music, or religious writing. Although this practice may be commonplace in the field of hospice and palliative medicine, no literature has been published about its perceived utility by team members with various competing demands on their time. OBJECTIVE: This study's objective was to obtain a better understanding about the perception and utility of a Thought for the Day held by clinicians rounding on an academic palliative medicine consult service. METHODS: A survey, containing qualitative and quantitative elements was sent to faculty, staff, and learners who participated in a Thought for the Day over the 18 months between March 2013 and October 2014. Twenty-eight responses were returned and analyzed. RESULTS: Most participants (23 of the 28) felt that the Thought for the Day was an important use of their time on the academic consult service. Differences were seen by gender and team role. Additionally, it was reported that the Thought for the Day improved the participants' perception of teamwork. CONCLUSION: The use of a Thought for the Day reflection may be beneficial and constructive even for a busy academic consult service.

Dexmedetomidine as adjuvant therapy for acute postoperative neuropathic pain crisis.

BACKGROUND: Dexmedetomidine is a potent α2-adrenergic agonist U.S. Food and Drug Administration (FDA) approved for sedation. While its use as an analgesic has been described in the palliative medicine literature, its use for managing an acute neuropathic pain episode is less well known. METHODS: Here we describe the use of adjuvant dexmedetomidine in a patient with metastatic sarcoma suffering from an acute postoperative neuropathic pain crisis. CONCLUSION: Among patients with acute neuropathic pain for whom additional opioids raises respiratory-related concerns, the use of dexmedetomidine should be considered as a viable treatment alternative.

A 768-lane microfabricated system for high-throughput DNA sequencing.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32x" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256x Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.