Healthcare team resilience during COVID-19: a qualitative study.

BACKGROUND: Resilience, in the field of Resilience Engineering, has been identified as the ability to maintain the safety and the performance of healthcare systems and is aligned with the resilience potentials of anticipation, monitoring, adaptation, and learning. In early 2020, the COVID-19 pandemic challenged the resilience of US healthcare systems due to the lack of equipment, supply interruptions, and a shortage of personnel. The purpose of this qualitative research was to describe resilience in the healthcare team during the COVID-19 pandemic with the healthcare team situated as a cognizant, singular source of knowledge and defined by its collective identity, purpose, competence, and actions, versus the resilience of an individual or an organization. METHODS: We developed a descriptive model which considered the healthcare team as a unified cognizant entity within a system designed for safe patient care. This model combined elements from the Patient Systems Engineering Initiative for Patient Safety (SEIPS) and the Advanced Team Decision Making (ADTM) models. Using a qualitative descriptive design and guided by our adapted model, we conducted individual interviews with healthcare team members across the United States. Data were analyzed using thematic analysis and extracted codes were organized within the adapted model framework. RESULTS: Five themes were identified from the interviews with acute care professionals across the US (N = 22): teamwork in a pressure cooker, consistent with working in a high stress environment; healthcare team cohesion, applying past lessons to present challenges, congruent with transferring past skills to current situations; knowledge gaps, and altruistic behaviors, aligned with sense of duty and personal responsibility to the team. Participants' described how their ability to adapt to their environment was negatively impacted by uncertainty, inconsistent communication of information, and emotions of anxiety, fear, frustration, and stress. Cohesion with co-workers, transferability of skills, and altruistic behavior enhanced healthcare team performance. CONCLUSION: Working within the extreme unprecedented circumstances of COVID-19 affected the ability of the healthcare team to anticipate and adapt to the rapidly changing environment. Both team cohesion and altruistic behavior promoted resilience. Our research contributes to a growing understanding of the importance of resilience in the healthcare team. And provides a bridge between individual and organizational resilience.

Re-Aiming Equity Evaluation in Clinical Decision Support: A Scoping Review of Equity Assessments in Surgical Decision Support Systems.

OBJECTIVE: We critically evaluated the surgical literature to explore the prevalence and describe how equity assessments occur when using clinical decision support systems. BACKGROUND: Clinical decision support (CDS) systems are increasingly used to facilitate surgical care delivery. Despite formal recommendations to do so, equity evaluations are not routinely performed on CDS systems and underrepresented populations are at risk of harm and further health disparities. We explored surgical literature to determine frequency and rigor of CDS equity assessments and offer recommendations to improve CDS equity by appending existing frameworks. METHODS: We performed a scoping review up to Augus 25, 2021 using PubMed and Google Scholar for the following search terms: clinical decision support, implementation, RE-AIM, Proctor, Proctor's framework, equity, trauma, surgery, surgical. We identified 1415 citations and 229 abstracts met criteria for review. A total of 84 underwent full review after 145 were excluded if they did not assess outcomes of an electronic CDS tool or have a surgical use case. RESULTS: Only 6% (5/84) of surgical CDS systems reported equity analyses, suggesting that current methods for optimizing equity in surgical CDS are inadequate. We propose revising the RE-AIM framework to include an Equity element (RE 2 -AIM) specifying that CDS foundational analyses and algorithms are performed or trained on balanced datasets with sociodemographic characteristics that accurately represent the CDS target population and are assessed by sensitivity analyses focused on vulnerable subpopulations. CONCLUSION: Current surgical CDS literature reports little with respect to equity. Revising the RE-AIM framework to include an Equity element (RE 2 -AIM) promotes the development and implementation of CDS systems that, at minimum, do not worsen healthcare disparities and possibly improve their generalizability.

Artificial Intelligence-enabled Decision Support in Surgery: State-of-the-art and Future Directions.

OBJECTIVE: To summarize state-of-the-art artificial intelligence-enabled decision support in surgery and to quantify deficiencies in scientific rigor and reporting. BACKGROUND: To positively affect surgical care, decision-support models must exceed current reporting guideline requirements by performing external and real-time validation, enrolling adequate sample sizes, reporting model precision, assessing performance across vulnerable populations, and achieving clinical implementation; the degree to which published models meet these criteria is unknown. METHODS: Embase, PubMed, and MEDLINE databases were searched from their inception to September 21, 2022 for articles describing artificial intelligence-enabled decision support in surgery that uses preoperative or intraoperative data elements to predict complications within 90 days of surgery. Scientific rigor and reporting criteria were assessed and reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines. RESULTS: Sample size ranged from 163-2,882,526, with 8/36 articles (22.2%) featuring sample sizes of less than 2000; 7 of these 8 articles (87.5%) had below-average (<0.83) area under the receiver operating characteristic or accuracy. Overall, 29 articles (80.6%) performed internal validation only, 5 (13.8%) performed external validation, and 2 (5.6%) performed real-time validation. Twenty-three articles (63.9%) reported precision. No articles reported performance across sociodemographic categories. Thirteen articles (36.1%) presented a framework that could be used for clinical implementation; none assessed clinical implementation efficacy. CONCLUSIONS: Artificial intelligence-enabled decision support in surgery is limited by reliance on internal validation, small sample sizes that risk overfitting and sacrifice predictive performance, and failure to report confidence intervals, precision, equity analyses, and clinical implementation. Researchers should strive to improve scientific quality.

A Randomized Trial Comparing Antibiotics with Appendectomy for Appendicitis.

BACKGROUND: Antibiotic therapy has been proposed as an alternative to surgery for the treatment of appendicitis. METHODS: We conducted a pragmatic, nonblinded, noninferiority, randomized trial comparing antibiotic therapy (10-day course) with appendectomy in patients with appendicitis at 25 U.S. centers. The primary outcome was 30-day health status, as assessed with the European Quality of Life-5 Dimensions (EQ-5D) questionnaire (scores range from 0 to 1, with higher scores indicating better health status; noninferiority margin, 0.05 points). Secondary outcomes included appendectomy in the antibiotics group and complications through 90 days; analyses were prespecified in subgroups defined according to the presence or absence of an appendicolith. RESULTS: In total, 1552 adults (414 with an appendicolith) underwent randomization; 776 were assigned to receive antibiotics (47% of whom were not hospitalized for the index treatment) and 776 to undergo appendectomy (96% of whom underwent a laparoscopic procedure). Antibiotics were noninferior to appendectomy on the basis of 30-day EQ-5D scores (mean difference, 0.01 points; 95% confidence interval [CI], -0.001 to 0.03). In the antibiotics group, 29% had undergone appendectomy by 90 days, including 41% of those with an appendicolith and 25% of those without an appendicolith. Complications were more common in the antibiotics group than in the appendectomy group (8.1 vs. 3.5 per 100 participants; rate ratio, 2.28; 95% CI, 1.30 to 3.98); the higher rate in the antibiotics group could be attributed to those with an appendicolith (20.2 vs. 3.6 per 100 participants; rate ratio, 5.69; 95% CI, 2.11 to 15.38) and not to those without an appendicolith (3.7 vs. 3.5 per 100 participants; rate ratio, 1.05; 95% CI, 0.45 to 2.43). The rate of serious adverse events was 4.0 per 100 participants in the antibiotics group and 3.0 per 100 participants in the appendectomy group (rate ratio, 1.29; 95% CI, 0.67 to 2.50). CONCLUSIONS: For the treatment of appendicitis, antibiotics were noninferior to appendectomy on the basis of results of a standard health-status measure. In the antibiotics group, nearly 3 in 10 participants had undergone appendectomy by 90 days. Participants with an appendicolith were at a higher risk for appendectomy and for complications than those without an appendicolith. (Funded by the Patient-Centered Outcomes Research Institute; CODA ClinicalTrials.gov number, NCT02800785.).

Surgery and the Smartphone: Can Technology Improve Equitable Access to Surgical Care?

Unfortunately, many patients in the United States experience disparities in access to surgical care, including geographic constraints, limited transportation and time, and financial hardships. Living in a "surgical care desert" results in a delay in care, driving up health care costs and reducing quality of care. In the age of COVID-19, patient access to health care has been further diminished by physical distancing guidelines, naturally increasing the need for innovative telehealth solutions. In this review, we focus on using smartphones for mobile health technology (mHealth) in the delivery of surgical care. This study is aimed at a general surgical audience that may be interested in exploring how mHealth can improve both access and health care quality for surgical patients and their families. We review the current uses of mHealth by surgeons for surgical site infection, new models of the perioperative surgical home, acute care surgical triage, remote patient monitoring devices, and evaluation and management of surgical consultations in the patient's home. We also review institutional and governmental barriers to the adoption of mHealth and offer some preliminary solutions that may aid the surgeon who wishes to implement this technology in their day-to-day practice.

Efficacy of a Breastfeeding Pain Self-Management Intervention: A Pilot Randomized Controlled Trial.

BACKGROUND: Over 90% of women experience pain during breastfeeding initiation and lack strategies to self-manage breast and nipple pain. Guided by the Individual and Family Self-Management Theory, a breastfeeding self-management (BSM) intervention targeted women's knowledge, beliefs, and social facilitation to manage their breast and nipple pain and achieve their breastfeeding goals. OBJECTIVES: The purpose of this longitudinal pilot randomized control trial (RCT) was to test the preliminary efficacy of the BSM intervention on general and specific pain related to breastfeeding. METHODS: Sixty women intending to breastfeed were approached within 48 hours of delivery to participate in this pilot RCT (30 randomized to the BSM intervention and 30 randomized to the control group). All participants provided baseline data before discharge and pain and breastfeeding measures at 1, 2, and 6 weeks. Participants in the BSM intervention group received educational modules addressing breast and nipple pain and biweekly, text-based nurse coaching and completed a daily breastfeeding journal. RESULTS: Women in the BSM intervention group reported significantly less breast and nipple pain at 1 and 2 weeks using a visual analog scale (p < .014 and p < .006) and at 2 weeks using the Brief Pain Inventory intensity scale (p < .029), but no difference in breastfeeding duration. DISCUSSION: The BSM intervention pilot demonstrates a positive effect on breastfeeding specific and overall generalized pain. Future investigation is needed to identify at-risk women of ongoing breastfeeding pain and develop precision interventions to sustain this beneficial health behavior for mothers and infants.

Promoting self-management of breast and nipple pain in breastfeeding women: Protocol of a pilot randomized controlled trial.

The majority of women experience pain during breastfeeding initiation with few strategies to manage breast and nipple pain. In fact, women cite breast and nipple pain as among the most common reasons for breastfeeding cessation. To address this important issue, we developed a breastfeeding self-management (BSM) intervention, based on the Individual and Family Self-Management Theory Framework. In this framework, self-management is conceptualized as a process in which women use knowledge, beliefs, and social facilitation to achieve breastfeeding goals. The purpose of this longitudinal pilot randomized controlled trial was to test the feasibility, acceptability, and preliminary efficacy of the BSM intervention with women initiating breastfeeding. Recruitment of 60 women intending to breastfeed occurred within 48 hr of delivery and women were randomized to either the intervention or usual care group. The BSM intervention group received BSM education modules that included information of how to manage breast and nipple pain and self-management support through biweekly texting from the study nurse, and were asked to complete a daily breastfeeding journal. Primary outcomes measured at baseline, 1, 2, and 6 weeks will be used to (a) evaluate feasibility, acceptability, and preliminary efficacy of the BSM intervention, and (b) assess the influence of protective and risk factors of breastfeeding pain (including individual genetic polymorphisms related to pain sensitivity) on process variables for self-management of breastfeeding and breastfeeding pain, and on proximal (breastfeeding pain severity and interference, breastfeeding frequency) and distal outcomes (breastfeeding exclusivity and duration and general well-being).

Wound Care Follow-Up From the Emergency Department Using a Mobile Application: A Pilot Study.

BACKGROUND: Many patients presenting to emergency departments (EDs) do not have primary care and risk being lost to follow-up. Technology has been used successfully in surgical populations for wound care follow-up yet this is not well studied in ED populations. OBJECTIVE: We aimed to conduct a pilot study demonstrating "smartphone" application-based follow-up after wound care in the ED. METHODS: We enrolled participants in 2 urban EDs using a smartphone application called Mobile Post-Operative Wound Evaluator (mPOWEr) and defined participation as photographic submission at any time during the study period. We collected demographic data, frequency of use of mPOWEr, number of photographs uploaded, and timing of uploads. RESULTS: We approached patients for study enrollment, and 67 patients (28%) were not enrolled because they had no access to a smartphone. Seventy-one patients (30%) declined to enroll, leaving 100 (42%) successfully enrolled. Smartphone ownership was more common among patients <40 years of age (81% vs. 64%, p = 0.004), more common among white patients than nonwhite patients (75% vs. 15%, p = 0.046), more common among patients approached at the university medical center than the trauma center (84% vs. 66%, p = 0.003), and among patients with commercial or other insurance than those with Medicare or Medicaid (92% vs. 54%, p < 0.001). Of those enrolled, 58% submitted a photograph. CONCLUSIONS: Patients presenting for wound care to the ED will participate in smartphone-based app communication for wound care follow-up and are satisfied with this option. Disparities in smartphone access must be considered when using this follow-up method.

The Medico-Legal 'Making' of 'The Transgender Child'.

Thirty years ago, the transgender child would have made no sense to the general public, nor to young people. Today, children and adolescents declare themselves transgender, the National Health Service diagnoses 'gender dysphoria', and laws and policy are developed which uphold young people's 'choice' to transition and to authorize stages at which medical intervention is permissible and desirable. The figure of the 'transgender child' presumed by medicine and law is not a naturally occurring category of person external to medical diagnosis and legal protection. Medicine and law construct the 'transgender child' rather than that the 'transgender child' exists independently of medico-legal discourse. The ethical issue of whether the child and young person can 'consent' to social and medical transition goes beyond legal assessment of whether a person under16 years has the mental capacity to consent, understand to what s/he is consenting, and can express independent wishes. It shifts to examination of the recent making of 'the transgender child' through the complex of power/knowledge/ethics of medicine and the law of which the child can have no knowledge but within which its own desires are both constrained and incited.

Trial of short-course antimicrobial therapy for intraabdominal infection.

BACKGROUND: The successful treatment of intraabdominal infection requires a combination of anatomical source control and antibiotics. The appropriate duration of antimicrobial therapy remains unclear. METHODS: We randomly assigned 518 patients with complicated intraabdominal infection and adequate source control to receive antibiotics until 2 days after the resolution of fever, leukocytosis, and ileus, with a maximum of 10 days of therapy (control group), or to receive a fixed course of antibiotics (experimental group) for 4±1 calendar days. The primary outcome was a composite of surgical-site infection, recurrent intraabdominal infection, or death within 30 days after the index source-control procedure, according to treatment group. Secondary outcomes included the duration of therapy and rates of subsequent infections. RESULTS: Surgical-site infection, recurrent intraabdominal infection, or death occurred in 56 of 257 patients in the experimental group (21.8%), as compared with 58 of 260 patients in the control group (22.3%) (absolute difference, -0.5 percentage point; 95% confidence interval [CI], -7.0 to 8.0; P=0.92). The median duration of antibiotic therapy was 4.0 days (interquartile range, 4.0 to 5.0) in the experimental group, as compared with 8.0 days (interquartile range, 5.0 to 10.0) in the control group (absolute difference, -4.0 days; 95% CI, -4.7 to -3.3; P<0.001). No significant between-group differences were found in the individual rates of the components of the primary outcome or in other secondary outcomes. CONCLUSIONS: In patients with intraabdominal infections who had undergone an adequate source-control procedure, the outcomes after fixed-duration antibiotic therapy (approximately 4 days) were similar to those after a longer course of antibiotics (approximately 8 days) that extended until after the resolution of physiological abnormalities. (Funded by the National Institutes of Health; STOP-IT ClinicalTrials.gov number, NCT00657566.).

The trophic life cycle stage of Pneumocystis species induces protective adaptive responses without inflammation-mediated progression to pneumonia.

Pneumocystis species are fungal pathogens that cause pneumonia in immunocompromised hosts. Lung damage during Pneumocystis pneumonia is predominately due to the inflammatory immune response. Pneumocystis species have a biphasic life cycle. Optimal innate immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, the trophic life cycle stage broadly suppresses proinflammatory responses to multiple pathogen-associated molecular patterns (PAMPs), including ß-1,3-glucan. Little is known about the contribution of these life cycle stages to the development of protective adaptive responses to Pneumocystis infection. Here we report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B-cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised recombination-activating gene 2 knockout (RAG2-/-) mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia.

The Trophic Life Cycle Stage of the Opportunistic Fungal Pathogen Pneumocystis murina Hinders the Ability of Dendritic Cells To Stimulate CD4+ T Cell Responses.

The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including ß-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with ß-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells.

Impact of Intravenous Immunoglobulin on Survival in Necrotizing Fasciitis With Vasopressor-Dependent Shock: A Propensity Score-Matched Analysis From 130 US Hospitals.

BACKGROUND: Shock frequently complicates necrotizing fasciitis (NF) caused by group A Streptococcus (GAS) or Staphylococcus aureus. Intravenous immunoglobulin (IVIG) is sometimes administered for presumptive toxic shock syndrome (TSS), but its frequency of use and efficacy are unclear. METHODS: Adult patients with NF and vasopressor-dependent shock undergoing surgical debridement from 2010 to 2014 were identified at 130 US hospitals. IVIG cases were propensity-matched and risk-adjusted. The primary outcome was in-hospital mortality and the secondary outcome was median length of stay (LOS). RESULTS: Of 4127 cases of debrided NF with shock at 121 centers, only 164 patients (4%) at 61 centers received IVIG. IVIG subjects were younger with lower comorbidity indices, but higher illness severity. Clindamycin and vasopressor intensity were higher among IVIG cases, as was coding for TSS and GAS. In-hospital mortality did not differ between matched IVIG and non-IVIG groups (crude mortality, 27.3% vs 23.6%; adjusted odds ratio, 1.00 [95% confidence interval, .55-1.83]; P = .99). Early IVIG (≤2 days) did not alter this effect (P = .99). Among patients coded for TSS, GAS, and/or S. aureus, IVIG use was still unusual (59/868 [6.8%]) and lacked benefit (P = .63). Median LOS was similar between IVIG and non-IVIG groups (26 [13-49] vs 26 [11-43]; P = .84). Positive predictive values for identifying true NF and debridement among IVIG cases using our algorithms were 97% and 89%, respectively, based on records review at 4 hospitals. CONCLUSIONS: Adjunctive IVIG was administered infrequently in NF with shock and had no apparent impact on mortality or hospital LOS beyond that achieved with debridement and antibiotics.

Outcomes in necrotizing soft tissue infections treated with therapeutic plasma exchange.

BACKGROUND: Treatment of necrotizing soft tissue infections (NSTIs) includes prompt surgical debridement and antibiotics, but despite standard care, the morbidity and mortality remain high. Since therapeutic plasma exchange (TPE) has been considered for treatment of severe sepsis, this study evaluates the efficacy of TPE for patients with NSTI. STUDY DESIGN AND METHODS: This is a retrospective study of patients with diagnosis of NSTI who received treatment with and without TPE over an 11-year period. The primary outcome was in-hospital mortality. RESULTS: Fifty-two patients with NSTI treated with TPE (TPE group) and 125 patients with NSTI not treated with TPE (non-TPE group) were assessed. Nineteen (36.5%) patients died in the TPE group, and 35 (28%) patients died in the non-TPE group. Within the TPE group, there was significant improvement in white blood cell (WBC) count and sodium levels 7 days after TPE treatment, but no improvement in creatinine. Inverse probability weighting based on propensity scores was used to compare survival in the TPE and non-TPE groups and demonstrated that TPE was associated with an increased odds of death (odds ratio, 2.8). A second analysis matched for six variables yielded 31 pairs and demonstrated no significant difference in mortality or length of stay. CONCLUSIONS: This study describes the largest series of patients with NSTIs treated with TPE and showed no evidence of clinical benefit. Further carefully designed studies with meaningful clinical endpoints would prove useful in assessing reproducibility and determining if there is a role for TPE in other forms of severe sepsis.

Prognostics of surgical site infections using dynamic health data.

Surgical Site Infection (SSI) is a national priority in healthcare research. Much research attention has been attracted to develop better SSI risk prediction models. However, most of the existing SSI risk prediction models are built on static risk factors such as comorbidities and operative factors. In this paper, we investigate the use of the dynamic wound data for SSI risk prediction. There have been emerging mobile health (mHealth) tools that can closely monitor the patients and generate continuous measurements of many wound-related variables and other evolving clinical variables. Since existing prediction models of SSI have quite limited capacity to utilize the evolving clinical data, we develop the corresponding solution to equip these mHealth tools with decision-making capabilities for SSI prediction with a seamless assembly of several machine learning models to tackle the analytic challenges arising from the spatial-temporal data. The basic idea is to exploit the low-rank property of the spatial-temporal data via the bilinear formulation, and further enhance it with automatic missing data imputation by the matrix completion technique. We derive efficient optimization algorithms to implement these models and demonstrate the superior performances of our new predictive model on a real-world dataset of SSI, compared to a range of state-of-the-art methods.

CHI: A contemporaneous health index for degenerative disease monitoring using longitudinal measurements.

In this paper, we develop a novel formulation for contemporaneous patient risk monitoring by exploiting the emerging data-rich environment in many healthcare applications, where an abundance of longitudinal data that reflect the degeneration of the health condition can be continuously collected. Our objective, and the developed formulation, is fundamentally different from many existing risk score models for different healthcare applications, which mostly focus on predicting the likelihood of a certain outcome at a pre-specified time. Rather, our formulation translates multivariate longitudinal measurements into a contemporaneous health index (CHI) that captures patient condition changes over the course of progression. Another significant feature of our formulation is that, CHI can be estimated with or without label information, different from other risk score models strictly based on supervised learning. To develop this formulation, we focus on the degenerative disease conditions, for which we could utilize the monotonic progression characteristic (either towards disease or recovery) to learn CHI. Such a domain knowledge leads us to a novel learning formulation, and on top of that, we further generalize this formulation with a capacity to incorporate label information if available. We further develop algorithms to mitigate the challenges associated with the nonsmooth convex optimization problem by first identifying its dual reformulation as a constrained smooth optimization problem, and then, using the block coordinate descent algorithm to iteratively solve the optimization with a derived efficient projection at each iteration. Extensive numerical studies are performed on both synthetic datasets and real-world applications on Alzheimer's disease and Surgical Site Infection, which demonstrate the utility and efficacy of the proposed method on degenerative conditions that include a wide range of applications.

The life cycle stages of Pneumocystis murina have opposing effects on the immune response to this opportunistic, fungal pathogen.

The cyst cell wall ß-glucans of Pneumocystis have been shown to stimulate immune responses in lung epithelial cells, dendritic cells, and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with these innate immune cells. Here, we report differences in the responses of both neonatal and adult mice to the trophic and cystic life cycle stages of Pneumocystis murina The adult and neonatal immune responses to infection with Pneumocystis murina trophic forms were less robust than the response to infection with a physiologically normal mixture of cysts and trophic forms. Cysts promoted the recruitment of nonresident innate immune cells and T and B cells into the lungs. Cysts, but not trophic forms, stimulated increased IFN-γ cytokine concentrations in the alveolar spaces, and an increase in IFN-γ-producing CD4+ T cells. In vitro, bone marrow-derived dendritic cells (BMDCs) stimulated with cysts produced the proinflammatory cytokines IL-1ß and IL-6. In contrast, trophic forms suppressed ß-glucan-, LTA-, and LPS-induced IL-1ß, IL-6, and TNFα production by BMDCs and antigen presentation to CD4+ T cells. The negative effects of trophic forms were not due to ligation of mannose receptor. Our results indicate that optimal innate and adaptive immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, trophic forms suppress ß-glucan-induced proinflammatory responses in vitro, suggesting that the trophic forms dampen cyst-induced inflammation in vivo.

Effects of Salt on the Stability of a G-Quadruplex from the Human c-MYC Promoter.

In an atmosphere of potassium ions, a modified c-MYC NHE III1 sequence with two G-to-T mutations (MYC22-G14T/G23T) forms a highly stable parallel-stranded G-quadruplex. The G-quadruplex exhibits a steady increase in its melting temperature, T(M), with an increase in the concentration of the stabilizing cation K(+). On the other hand, an increase in the concentration of nonstabilizing Cs(+) or TMA(+) cations at a constant concentration of K(+) causes a sharp decline in T(M) followed by a leveling off at ∼200 mM Cs(+) or TMA(+). At 51 °C and 600 µM K(+), an increase in Cs(+) concentration from 0 to 800 mM leads to a complete unfolding of the G-quadruplex. These observations are consistent with the picture in which more counterions accumulate in the vicinity of the unfolded state of MYC22-G14T/G23T (nonspecific ion binding) than in that of the G-quadruplex state. We estimate that the unfolded state condenses one extra counterion compared to the G-quadruplex state. Taken together with our earlier results, our data suggest that sodium or potassium cations sequestered inside the central cavity stabilize the G-quadruplex conformation acting as specifically bound ligands. Nonspecifically bound (condensed) counterions may slightly stabilize, exert no influence (human telomeric G-quadruplexes), or strongly destabilize (MYC22-G14T/G23T) the G-quadruplex conformation. We offer a structural rationalization for the enhanced thermal stability of the MYC22-G14T/G23T G-quadruplex.