The Tween Years: Thriving as a Mid-Career Academic Hospice and Palliative Medicine Clinician.

In the young and rapidly evolving field of hospice and palliative medicine (HPM), the transition from early to mid-career can be a precarious time. The high rates of burnout and low rates of work-life balance and satisfaction found in mid-career jeopardize our field's ability to maintain a healthy workforce. In this series, we present three cases that highlight common issues encountered during the early to mid-career transition in academic HPM and present several strategies for navigating challenges. A web of mentors/connections, academic map, and continuing education to enhance teaching skills are several concrete tools explored. To sustain a robust HPM workforce, such practical and structured supports during the particularly challenging mid-career transition are crucial.

Understanding Influence within the Context of Nursing: Development of the Adams Influence Model Using Practice, Research, and Theory.

Acquiring influence, and knowing how to use it, is a required competency for nurse leaders, yet the concept of influence and how it works is not well described in the nursing literature. In this article, the authors examine what is known about influence and present an influence model specific to nurse leaders. The Adams Influence Model was developed through an iterative process and is based on a comprehensive review of the influence literature, expert commentary, multiple pilot studies, evaluation of nursing theories, and validation by an external data source. Rather than defining "how to" influence, the model serves as a guide for personal reflection, helping nurse leaders understand and reflect on the influence process and factors, tactics, and strategies they can use when seeking to influence others.

Reduction of eotaxin production and eosinophil recruitment by pulmonary autologous macrophage transfer in a cockroach allergen-induced asthma model.

We sought to investigate the effects of cockroach allergen (CRA) exposure on the lung macrophage population to determine how different macrophage phenotypes influence exacerbation of disease. CRA exposure caused significantly reduced expression of CD86 on lung macrophages. These effects were not systemic, as peritoneal macrophage CD86 expression was not altered. To investigate whether naïve macrophages could reduce asthma-like pulmonary inflammation, autologous peritoneal macrophages were instilled into the airways 24 h before the final CRA challenge. Pulmonary inflammation was assessed by measurement of airway hyperresponsiveness, mucin production, inflammatory cell recruitment, and cytokine production. Cell transfer did not have significant effects in control mice, nor did it affect pulmonary mucin production or airway hyperresponsiveness in control or CRA-exposed mice. However, there was significant reduction in the number of eosinophils recovered in the bronchoalveolar lavage (BAL) (5.8 × 105 vs. 0.88 × 105), and total cell recruitment to the airways of CRA-exposed mice was markedly reduced (1.1 × 106 vs. 0.57 × 106). The reduced eosinophil recruitment was reflected by substantially lower levels of eosinophil peroxidase in the lung and significantly lower concentrations of eotaxins in BAL (eotaxin 1: 3 pg/ml vs. undetectable; eotaxin 2: 2,383 vs. 131 pg/ml) and lung homogenate (eotaxin 1: 1,043 vs. 218 pg/ml; eotaxin 2: 10 vs. 1.5 ng/ml). We conclude that CRA decreases lung macrophage CD86 expression. Furthermore, supplementation of the lung cell population with peritoneal macrophages inhibits eosinophil recruitment, achieved through reduction of eotaxin production. These data demonstrate that transfer of naïve macrophages will reduce some aspects of asthma-like pulmonary inflammation in response to CRA.

ELISA rescue protocol: recovery of sample concentrations from an assay with an unsuccessful standard curve.

The Enzyme-linked Immunosorbant Assay (ELISA) is a method commonly used to measure proteins in various biological matrices, due to its ease of performance and relatively low cost. In order for quantitative data to be generated, a reference standard curve must be prepared for each assay; however, due to investigator error or standard protein degradation, otherwise representative experimental sample data are rendered useless. Herein, we describe a protocol by which sample concentrations can be recovered from assays in which the standard curve fails. The ΔOD values of the experimental samples are used to generate a new standard curve, which is applied back to the original plate. For validation of this method, experimental sample concentrations obtained using acceptable standard curves were potted against those calculated using this new method. Using linear regression analysis, we show a near 1:1 correlation between sample concentrations, with r(2) values between 0.98 and 0.99 and slopes between 0.97 and 1.10. This method demonstrates that assays resulting in unusable standard curves do not require re-assay of all samples. Instead, the experimental sample concentrations can be retrieved saving the investigator the time and resources required to rerun samples or repeat entire experiments.

Cockroach allergens induce biphasic asthma-like pulmonary inflammation in outbred mice.

OBJECTIVES: The aim of this study is to define the kinetics of the pulmonary inflammatory response in cockroach allergen (CRA) sensitized and challenged outbred mice. METHODS: Asthma-like pulmonary inflammation was induced with three pulmonary exposures to CRA, without the use of adjuvants. Mice were sacrificed at multiple time points and asthma-like pulmonary inflammation quantified. RESULTS: Several pulmonary parameters showed a pronounced biphasic inflammatory response with an early stage (1.5 hours post challenge) and late stage (24 hours). The initial phase was characterized by the production of multiple inflammatory mediators, including CXC chemokines, and the recruitment of neutrophils to the lung. The number of pulmonary eosinophils decreased in the early phase but quickly rebounded. Both the early and late phases had increases in TNF production in addition to airways hyperreactivity. The model also demonstrated early production of mucin with clearance by 12 hours followed by new accumulation of mucin in the pulmonary epithelial cells. Eotaxins within the lung peaked at about 12 hours and the numbers of eosinophils in the lung remained constant throughout the 48 hours of the study. CONCLUSIONS: The pulmonary inflammatory parameters in response to a clinically relevant allergen define a biphasic response. These data may be used to investigate the pathogenesis of the disease and develop targeted therapies for the distinct phases.

Pulmonary endotoxin tolerance protects against cockroach allergen-induced asthma-like inflammation in a mouse model.

BACKGROUND: Compounds which activate the innate immune system, such as lipopolysaccharide, are significant components of ambient air, and extremely difficult to remove from the environment. It is currently unclear how prior inhalation of endotoxin affects allergen sensitization. We examined whether lung-specific endotoxin tolerance induction prior to sensitization can modulate the response to allergen. METHODS: Endotoxin tolerance was induced by repeated intratracheal exposure to endotoxin. All mice were then sensitized and challenged by direct intratracheal instillation of cockroach allergen. RESULTS: After allergen sensitization and challenge, endotoxin tolerant mice had significantly decreased airways hyperresponsiveness to methacholine challenge, which was confirmed by invasive lung function tests. Decreased goblet cell hyperplasia and mucus production were also found by histological assessment. Tolerant mice were protected from airway eosinophilia through the mechanism of reduced CCL11 and CCL24. Interestingly, endotoxin tolerant mice had only a modest reduction in cockroach-specific IgE; however, total IgE was significantly reduced. CONCLUSIONS: These data show that induction of endotoxin tolerance prior to sensitization protects against the hallmark features of asthma-like inflammation, and that transient modulation of innate immunity can have long-lasting effects on adaptive responses.

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin-modifying HDACi exhibit anti-inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM-CSF-stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b(+)Ly6C(+)F4/80(int)CD115(+)), which suppressed allogeneic T cell proliferation in a NOS- and HO-1-dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin(-)Sca-1(+)c-Kit(+)). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM-CSF administration and increased the number of CD11b(+)Gr1(+) cells in BM and spleen. Increased numbers of Gr1(+) cells, which suppressed T cell proliferation, were recovered from spleens of TSA-treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti-inflammatory action of HDACi.

Diesel exhaust particulates exacerbate asthma-like inflammation by increasing CXC chemokines.

Particulate matter heavily pollutes the urban atmosphere, and several studies show a link between increased ambient particulate air pollution and exacerbation of pre-existing pulmonary diseases, including asthma. We investigated how diesel exhaust particulates (DEPs) aggravate asthma-like pulmonary inflammation in a mouse model of asthma induced by a house dust extract (HDE) containing cockroach allergens and endotoxin. BALB/c mice were exposed to three pulmonary challenges via hypopharyngeal administration of an HDE collected from the home of an asthmatic child. One hour before each pulmonary challenge, mice were exposed to DEP or PBS. Pulmonary inflammation was assessed by histological features, oxidative stress, respiratory physiological features, inflammatory cell recruitment, and local CXC chemokine production. To prove the role of CXC chemokines in the augmented inflammation, CXC chemokine-specific antibodies were delivered to the lungs before DEP exposure. DEP exacerbated HDE-induced airway inflammation, with increased airway mucus production, oxidative stress, inflammatory cell infiltration, bronchoalveolar lavage concentrations of CXC chemokines, and airway hyperreactivity. Neutralization of airway keratinocyte-derived chemokine and macrophage inflammatory protein-2 significantly improves the respiratory function in addition to decreasing the infiltration of neutrophils and eosinophils. Blocking the chemokines also decreased airway mucus production. These results demonstrate that DEP exacerbates airway inflammation induced by allergen through increased pulmonary expression of the CXC chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein-2).

Herbal medicine treatment reduces inflammation in a murine model of cockroach allergen-induced asthma.

BACKGROUND: Asthma is a significant disease among children, and its prevalence has increased notably during the last 2 decades. A traditional Korean medicine, So-Cheong-Ryong-Tang (SCRT), has been used for the treatment of asthma in Asia for centuries, but its mechanism for reducing bronchopulmonary inflammation in asthma has yet to be elucidated. OBJECTIVE: To investigate whether the herbal extract SCRT inhibits inflammation in a mouse model of cockroach allergen-induced asthma. METHODS: A house dust extract containing endotoxin and cockroach allergens was used for immunization and 2 additional pulmonary challenges in BALB/c mice. Mice were treated with SCRT or vehicle 1 hour before each pulmonary challenge. Respiratory parameters were evaluated by whole-body plethysmography and forced oscillation methods 24 hours after the last challenge. Bronchoalveolar lavage (BAL) fluid was collected, and histologic sections of lung were prepared either 4 or 24 hours after the last house dust extract challenge. RESULTS: SCRT treatment significantly reduced the hyperreactivity of the airways as measured by whole-body plethysmography and direct measurement of airway resistance. Inflammation was significantly inhibited by SCRT treatment as demonstrated by reduced plasma IgE levels and improved pulmonary histologic characteristics. SCRT significantly reduced the number of neutrophils in the BAL fluid and also significantly reduced the BAL levels of CXC chemokines, providing a potential mechanism for the reduced inflammation. In a similar fashion, SCRT reduced eosinophil recruitment and BAL levels of eotaxin and RANTES. CONCLUSION: These data indicate that SCRT treatment alleviates asthma-like pulmonary inflammation via suppression of specific chemokines.

Reducing LPS content in cockroach allergens increases pulmonary cytokine production without increasing inflammation: a randomized laboratory study.

BACKGROUND: Endotoxins are ubiquitously present in the environment and constitute a significant component of ambient air. These substances have been shown to modulate the allergic response, however a consensus has yet to be reached whether they attenuate or exacerbate asthmatic responses. The current investigation examined whether reducing the concentration of lipopolysaccharide (LPS) in a house dust extract (HDE) containing high concentrations of both cockroach allergens 1 and LPS would attenuate asthma-like pulmonary inflammation. METHODS: Mice were sensitized with CRA and challenged with the intact HDE, containing 182 ng of LPS, or an LPS-reduced HDE containing 3 ng LPS, but an equivalent amount of CRA. Multiple parameters of asthma-like pulmonary inflammation were measured. RESULTS: Compared to HDE challenged mice, the LPS-reduced HDE challenged mice had significantly reduced TNFα levels in the bronchoalveolar lavage fluid. Plasma levels of IgE and IgG1 were significantly reduced, however no change in CRA-specific IgE was detected. In HDE mice, plasma IgG2a levels were similar to naïve mice, while LPS-reduced HDE mice had significantly greater concentrations. Reduced levels of LPS in the HDE did not decrease eosinophil or neutrophil recruitment into the alveolar space. Equivalent inflammatory cell recruitment occurred despite having generally higher pulmonary concentrations of eotaxins and CXC chemokines in the LPS-reduced HDE group. LPS-reduced HDE challenge induced significantly higher concentrations of IFNγ, and IL-5 and IL-13 in the BAL fluid, but did not decrease airways hyperresponsiveness or airway resistance to methacholine challenge. CONCLUSION: These data show that reduction of LPS levels in the HDE does not significantly protect against the severity of asthma-like pulmonary inflammation.

Oral tolerance inhibits pulmonary eosinophilia in a cockroach allergen induced model of asthma: a randomized laboratory study.

BACKGROUND: Antigen desensitization through oral tolerance is becoming an increasingly attractive treatment option for allergic diseases. However, the mechanism(s) by which tolerization is achieved remain poorly defined. In this study we endeavored to induce oral tolerance to cockroach allergen (CRA: a complex mixture of insect components) in order to ameliorate asthma-like, allergic pulmonary inflammation. METHODS: We compared the pulmonary inflammation of mice which had received four CRA feedings prior to intratracheal allergen sensitization and challenge to mice fed PBS on the same time course. Respiratory parameters were assessed by whole body unrestrained plethysmography and mechanical ventilation with forced oscillation. Bronchoalveolar lavage fluid (BAL) and lung homogenate (LH) were assessed for cytokines and chemokines by ELISA. BAL inflammatory cells were also collected and examined by light microscopy. RESULTS: CRA feeding prior to allergen sensitization and challenge led to a significant improvement in respiratory health. Airways hyperreactivity measured indirectly via enhanced pause (Penh) was meaningfully reduced in the CRA-fed mice compared to the PBS fed mice (2.3 ± 0.4 vs 3.9 ± 0.6; p = 0.03). Directly measured airways resistance confirmed this trend when comparing the CRA-fed to the PBS-fed animals (2.97 ± 0.98 vs 4.95 ± 1.41). This effect was not due to reduced traditional inflammatory cell chemotactic factors, Th2 or other cytokines and chemokines. The mechanism of improved respiratory health in the tolerized mice was due to significantly reduced eosinophil numbers in the bronchoalveolar lavage fluid (43300 ± 11445 vs 158786 ± 38908; p = 0.007) and eosinophil specific peroxidase activity in the lung homogenate (0.59 ± 0.13 vs 1.19 ± 0.19; p = 0.017). The decreased eosinophilia was likely the result of increased IL-10 in the lung homogenate of the tolerized mice (6320 ± 354 ng/mL vs 5190 ± 404 ng/mL, p = 0.02). CONCLUSION: Our results show that oral tolerization to CRA can improve the respiratory health of experimental mice in a CRA-induced model of asthma-like pulmonary inflammation by reducing pulmonary eosinophilia.

Assessing pulmonary pathology by detailed examination of respiratory function.

Pulmonary inflammation causes multiple alterations within the lung, including mucus production, recruitment of inflammatory cells, and airway hyperreactivity (AHR). Measurement of AHR by direct, invasive means (eg, mechanical ventilation) or noninvasive techniques, like whole body plethysmography (WBP), assesses the severity of pulmonary inflammation in animal models of inflammatory lung disease. Direct measurement of AHR is acknowledged as the most accurate method for assessing airway mechanics, but analysis of all data obtained from WBP may offer insights into which inflammatory aspects of the lung are altered along with AHR. Using WBP, we compared the respiratory parameters of two groups of mice sensitized with cockroach allergen. One group was treated with dexamethasone (Dex) before final challenge (Dex-Asthma), while the other group received vehicle treatment (Asthma). Respiratory parameters from plethysmography revealed that Dex-Asthma mice compensated to maintain high minute ventilation, whereas Asthma mice showed significant impairment in minute ventilation despite increased peak expiratory flow (103 ± 5 ml/min vs. 69 ± 70 ml/min). The WBP data suggest that enhanced air exchange in the Dex-Asthma mice results from significant decreases in airway mucus production. Additional studies with quantitative morphometry of histological sections confirmed that Dex reduced airway mucus. In conclusion, a detailed examination of WBP parameters can accurately assess the respiratory health of mice and will help direct additional studies.

Tolerogenic dendritic cells and myeloid-derived suppressor cells: potential for regulation and therapy of liver auto- and alloimmunity.

Organ transplantation is now established as an accepted treatment for end-stage liver disease, acute fulminant hepatic liver failure and hepatocellular carcinoma. While early graft acceptance rates have increased markedly due to improved immunosuppressive drug regimens, rates of late graft failure remain largely unchanged. Recent findings suggest that in addition to alloimmunity, chronic rejection of liver allografts may also reflect de novo autoimmune hepatitis or recurrence of pre-existing hepatic autoimmune disease. Dendritic cell (DC)- based therapy is a promising experimental approach to promotion of transplant tolerance and the treatment of autoimmune diseases. Newly emerging evidence also demonstrates the potential efficacy of myeloid-derived suppressor cells (MDSC) in the antigen (Ag)-specific regulation of T-cell responses. Herein, we discuss current understanding of liver autoimmunity post-transplantation, along with current approaches for the development of tolerogenic DC, and the potential use of MDSC for the development of stable, Ag-specific tolerance.

Chronic pulmonary LPS tolerance induces selective immunosuppression while maintaining the neutrophilic response.

LPS challenge causes potent activation of innate immunity. Because LPS is ubiquitously present in ambient air, repeated inhalation may lead to activation of the pulmonary immune response. If this activation is unregulated, chronic LPS inhalation would lead to persistent inflammation and organ damage. We hypothesized that the lung uses the mechanism of LPS tolerance to maintain the balance between hypoinflammatory and hyperinflammatory states. We developed a model of chronic pulmonary LPS tolerance induced by pulmonary exposure to 1 microg LPS for 4 consecutive days. Mice were challenged with 10 microg of LPS 24 h later. TNF-alpha protein was significantly decreased in the bronchoalveolar lavage fluid of tolerant versus nontolerant mice, whereas IL-6 levels were significantly increased in the tolerant group. Tolerant mice were also protected from airway hyperresponsiveness. M2 and M3 muscarinic receptor mRNA was significantly decreased in the lungs of tolerant mice, suggesting a mechanism for the decreased airway hyperresponsiveness. CXCL2 was significantly reduced in tolerant mice, but CXCL1 was equivalent between groups. No difference was seen in neutrophil recruitment to the alveolar space. Interestingly, LPS tolerance does not confer cross-tolerance to the Toll-like receptor (TLR) 2 stimulus Pam3Cys. TNF-alpha and IL-6 concentrations were significantly increased in LPS-tolerant mice challenged with Pam3Cys; however, chemokine concentrations were unaffected. Our data show that repeated LPS inhalation results in differential regulation of cytokines but does not inhibit neutrophil recruitment. This unrestricted neutrophil recruitment may represent a mechanism by which individuals may be protected from pulmonary bacterial infection and pneumonia.

Inbred and outbred mice have equivalent variability in a cockroach allergen-induced model of asthma.

Outbred mice traditionally are considered to display high variability, thereby limiting their use in some studies. Researchers frequently are encouraged to use inbred strains of mice because of the greater homogeneity of these experimental animals. We compared the pulmonary inflammatory response of inbred BALB/cJ mice to that of outbred HSD-ICR mice by measuring multiple variables, including cytokines, chemokines, number of pulmonary inflammatory cells, and respiratory parameters. Cockroach allergens induced significant pulmonary inflammation in both BALB and ICR mice. Our comparisons of the coefficients of variance for 148 discrete data sets for each strain or stock indicated that BALB and ICR mice have roughly equivalent intrastrain or -stock variability in our model of asthma-like pulmonary inflammation. The average coefficient of variance, calculated as the ratio of the SD to the mean of a data set, was 0.35 ± 0.34 for BALB mice compared with 0.31 ± 0.32 for ICR mice. In conclusion, inbred BALB and outbred ICR mice have roughly equivalent intrastrain or -stock variability in a murine model of asthma-like pulmonary inflammation.

Acute pulmonary lipopolysaccharide tolerance decreases TNF-alpha without reducing neutrophil recruitment.

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-alpha. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-alpha were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-alpha mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-alpha was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-alpha expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-alpha levels, while still maintaining the antimicrobial capacity of the lung.

The ELISA Standard Save: calculation of sample concentrations in assays with a failed standard curve.

The enzyme-linked immunosorbent assay (ELISA) is often used to measure protein levels in plasma and other solutions. In order for the assay to be quantitative, a standard curve must be prepared for each assay. Technical blunders in preparing the standard curve can render otherwise representative sample values useless. In order to recover these data, a protocol has been developed whereby a new standard curve is generated using the DeltaOD values of the samples. This new standard is applied to the original plate in order to determine sample concentrations. To validate this method, sample concentrations obtained using an acceptable standard curve were plotted against those obtained using the new standard curve. Linear regression analysis showed a 1:1 correlation between concentrations, with r(2) values ranging from 0.98-0.99 and slopes ranging from 0.97-1.10. In this manner, data for these samples are preserved, saving the investigator the time and money involved in repeating experiments.