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Background: Cancer is one of the leading causes of death worldwide, and cardiopulmonary comorbidities may further adversely affect cancer prognosis. We recently described lung cancer-associated pulmonary hypertension (PH) as a new form of PH and comorbidity of lung cancer. While patients with lung cancer with PH had significantly reduced overall survival compared with patients without PH, the prevalence and impact of PH in other cancers remain unclear., Methods: In this retrospective, observational cohort study, we analysed the prevalence and impact of PH on clinical outcomes in 1184 patients with solid tumours other than lung cancer, that is, colorectal, head and neck, urological, breast or central nervous system tumours, using surrogate markers for PH determined by CT., Results: PH prevalence in this cohort was 10.98%. A Cox proportional hazard model revealed a significant reduction in the median survival time of patients with cancer with PH (837 vs 2074 days; p<0.001). However, there was no correlation between pulmonary metastases and PH. A subgroup analysis showed that PH was linked to decreased lung and cardiac function. Additionally, PH was associated with systemic arterial hypertension (p<0.001) and coronary artery disease (p=0.014), but not emphysema., Conclusions: In this study, fewer patients with cancer had surrogate parameters for PH compared with previously published results among patients with lung cancer. Consequently, the prevalence of PH in other cancers might be lower compared with lung cancer; however, PH still has a negative impact on prognosis. Furthermore, our data does not provide evidence that lung metastases cause PH. Thus, our results support the idea that lung cancer-associated PH represents a new category of PH. Our results also highlight the importance of further studies in the field of cardio-oncology., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: In Germany, numerous health care providers work in the field of bereavement care. An epidemiological study of the field immediately after the introduction of the ICD-11 diagnosis "Prolonged Grief Disorder" (PGD) is pending., Methods: The second survey of the trend study, which is designed over a total of three measurement points at intervals of one decade each, explores whether and to what extent the field of bereavement care has evolved after the introduction of the grief-specific diagnosis PGD. For this purpose, providers of bereavement care at various organizational levels were asked to participate in an online survey. The survey was conducted from October 2020 to January 2021., Results: 456 questionnaires were included in the study. Data analysis was descriptive. 80.5% of the participants have an additional grief-specific qualification, 59.4% base their work on a grief-specific concept. In view of the PGD diagnosis, only a few respondents have participated in a PGD-related advanced training. Most are afraid that the topic of grief will increasingly be subsumed in the medical-psychological-therapeutic field. An increase in bereavement research and the number and quality of continuing education is also expected., Discussion: Some positive trends in German bereavement care can be identified. However, there is a need for further development in the areas "designation of the activity", "diagnostics", and "intervention". Also, there is a need for training with regard to PGD., Conclusion: After the introduction of PGD, the field of bereavement care in Germany turns out to be slightly different. The diagnosis is viewed skeptically., (Copyright © 2023. Published by Elsevier GmbH.)

Background: There is a research gap regarding safety concerns of patients at the end of life. The aim of this study was, therefore, to explore whether patients under specialist community palliative care feel safe at home and which factors affect the perceived safety. Furthermore, we investigated if perception of safety is associated with different aspects of subsequent care., Methods: Using a standardized questionnaire, a cross-sectional survey was conducted among 100 specialist community palliative care patients. Logistic regression was used to examine the strength of the association between clinical and socio-demographic variables and the perception of safety. After a 6-month follow-up period, we analyzed differences in various care-related outcomes between patients with unaffected and impaired perceptions of safety., Results: In our study, one in five patients receiving specialist community palliative care expressed safety concerns. Subdomains of safety that were reported most frequently were physical disability (60%), physical symptoms (30%), psychological symptoms (26%), and side effects/complications of drug therapy (19%). Of the participants surveyed after the initial COVID-19 lockdown, 35.1% reported that they felt their safety had been adversely affected by the pandemic. Compromised safety perception was associated with higher levels of palliative care-related problems, and proximity to death., Conclusions: Our study uncovered relevant safety concerns of palliative care patients receiving specialist community palliative care. The insights gained into patient-reported problems may help healthcare professionals to identify situations where patients feel unsafe. Further research should address primary and secondary prevention measures to improve the quality of end-of-life care in the home environment.

Background: The coronavirus disease 2019 (COVID-19) pandemic has challenged health care systems worldwide. In Germany, patients in a palliative care setting have the opportunity to receive treatment by a specialised mobile outpatient palliative care team (OPC). The given retrospective single centre analysis describes the use of OPC structures for terminally ill COVID-19 patients during the height of the pandemic in Germany and aims to characterise this exceptional OPC patient collective., Methods: First, death certificates were analysed in order to collect data about the place of death of all deceased COVID-19 patients (n = 471) within our local governance district. Second, we investigated whether advance care planning structures were established in local nursing homes (n = 30) during the height of the COVID-19 pandemic in 2020. Third, we examined patient characteristics of COVID-19 negative (n = 1579) and COVID-19 positive (n = 28) patients treated by our tertiary care centre guided OPC service., Results: The analysis of death certificates in our local district revealed that only 2.1% of all deceased COVID-19 patients had succumbed at their home address (n = 10/471). In contrast, 34.0% of COVID-19 patients died in nursing homes (n = 160/471), whereas 63.5% died in an inpatient hospital setting (n = 299/471). A large proportion of these hospitalised patients died on non-intensive care unit wards (38.8%). Approximately 33.0% of surveyed nursing homes had a palliative care council service and 40.0% of them offered advance care planning (ACP) structures for their nursing home residents. In our two OPC collectives we observed significant differences concerning clinical characteristics such as the Index of Eastern Cooperative Oncology Group [ECOG] (p = 0.014), oncologic comorbidity (p = 0.004), as well as referrer and primary patient location (p = 0.001, p = 0.033)., Conclusions: Most COVID-19 patients in our governance district died in an inpatient setting. However, the highest number of COVID-19 patients in our governance district who died in an outpatient setting passed away in nursing homes where palliative care structures should be further expanded. COVID-19 patients who died under the care of our OPC service had considerably fewer oncologic comorbidities. Finally, to relieve conventional health care structures, we propose the expansion of established OPC structures for treating terminally ill COVID-19 patients., (© 2022. The Author(s).)

Purpose: The care of older neurosurgical patients at the end life is a particularly demanding challenge. Especially, the specific needs of very old patients with glioblastoma at the end of life are at risk of being deprived of adequate care., Methods: Based on a narrative literature review, this article aims to explore key issues of the thematic intersection of geriatric glioblastoma patients, palliative care and neurosurgery., Results and Discussion: Four key issues were identified: patient-centeredness (need orientation and decision making), early palliative care, advance care planning, and multi-professionalism. Possible benefits and barriers are highlighted with regard to integrating these concepts into neurosurgery., Conclusions: Palliative care complements neurosurgical care of geriatric glioblastoma multiforme patients to optimise care for this highly vulnerable category of patients., (© 2022. The Author(s).)

Grieving in high-speed society. An acceleration-focused temporal perspective Objectives: How long does it take the bereaved to cope with a loss of a significant other? Early findings assume weeks. Later studies speak of years. Nevertheless, the grace period for the bereaved in the modern world seems to be constantly shortening, despite objectively constant time, multiple time gains and savings. Methods: The process of acceleration described by Hartmut Rosa offers an explanation for this paradoxical disproportion. From a time-analytic perspective, it becomes clear that coping with loss runs counter to the dynamization of society. Results: Grief as a bio-psychological process cannot be accelerated as such. Instead of therapy-immanent solutions, changes are needed that are reflected on a societal level. Conclusions: The approaches 'grief literacy' and 'compassionate communities' can help to implement bereavement into the society as a whole.

Introduction: In Germany, many health care providers work in bereavement care. An epidemiological study of this field of work has not yet been conducted., Methods: In the initial survey of this three-phase trend study the situation of grief-specific health care in Germany in 2009/2010 is examined, i. e., at the time before the introduction of the new ICD-11 diagnosis of Prolonged Grief Disorder. For this purpose, bereavement care providers at different organizational levels participated in an online survey., Results: 410 questionnaires were included in the study. Data analysis was descriptive. The most frequent reason for using grief-specific support services was the loss of a partner. In more than half of all cases of bereavement, people experienced a loss that was preceded by an illness and suffering. More than half of those providing bereavement care do not follow a concept of intervention., Discussion: There is considerable need for further development in German bereavement care, in particular with regard to qualification and the degree of professionalization, designation of the respective interventions, diagnostics, and intervention., Conclusion: This three-phase trend study enables health care providers to derive bereavement care service standards that aim to treat people according to their needs. Whether the diagnosis of Prolonged Grief Disorder has led to changes in bereavement care is currently analyzed in the second survey phase of the study., (Copyright © 2022. Published by Elsevier GmbH.)

Introduction: Specialized palliative home care (SPHC) enables children and adolescents with life-limiting illnesses and complex needs to receive care at home. In addition to controlling symptoms and stabilizing the psychosocial situation, crisis anticipation is a component of SPHC. Since the establishment of the reporting SPHC team, parents have called for additional help from emergency medical services (EMS) in emergency situations with unexpected frequency. Children with life limiting diseases could undergo invasive procedures and unhelpful treatments with uncertain consequences. The questions arose as to which factors led to the involvement of the EMS in a palliative situation, what therapy was performed and what outcome could be reached. Methods: Records of the pediatric SPHC patients and EMS call-outs in these children of the reporting SPHC-team in the central region of Hesse, Germany (population: 1.1 million) were retrospectively analyzed from 01.11.2014 to 01.05.2021. The causes of the call-outs, the existence of an emergency agreement, the National Advisory Committee for Aeronautics (NACA) score, EMS therapy and outcome were examined. Patient data included age, palliative-justifying diagnosis, duration and intensity of care, place of death and median overall survival (MOS) and palliative SHPC treatment. Results: In total, 172 patients were analyzed during the study period. There were 27 EMS calls for a total of 20 patients/families (= EMS group). Palliative illness or a complication was the most frequent cause of call-outs. The patients in the EMS group were significantly less likely to have a DNR order, required more home visits and telephone calls and were under SPHC care for longer. There was a significantly higher proportion of crisis interventions at home visits. The children in the EMS group died less often from the underlying disease. Of the remaining 152 patients (= non-EMS group), a significantly higher proportion had a European home country. Conclusions: Despite the introduction of the SPHC, parents still call the EMS. Good cooperation and joint training should be sought to prepare all those involved for future call-outs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hauch, El Mohaui, Wolff, Vaillant, Brill, Schneck, Ströter, Sibelius, Kriwy and Berthold.)

Background: About 80% of all people in Germany die in inpatient care. Around every fifth person in inpatient care is relocated to another care area in the last phase of their life. That is more than 150,000 people being relocated, often without indication. 13 risk factors were identified for these non-indicated relocations., Method: With the support of the AWMF, two regionally effective guidelines were developed and implemented in a maximum care hospital and a care facility. A palliative consultation service has been established in the university hospital. Comprehensive personnel and organizational development was carried out in the care facility. Different collaborations with relevant regional partners of both model institutions were systematically expanded., Results and Conclusions: The relocations could be significantly reduced despite the short duration of the project. This was also possible through the establishment of decision-making aids and digital implementation support. The results of the accompanying ethical and social research justify the procedure: There is an increase in the satisfaction of relatives and employees., (© 2021. Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Context: Open end-of-life communication is especially important within the patient-family unit of care and can positively affect their medical, psychological, and relational outcomes. Nevertheless, end-of-life discussions are often perceived as difficult and avoided., Objectives: To develop and validate the Difficulties in End-of-Life Discussions - Family Inventory (DEOLD-FI) to allow a systematic assessment of reasons why people shy away from end-of-life discussions., Methods: Patients with advanced cancer were recruited and completed the DEOLD-FI and measures of avoidance of cancer communication, quality of life and distress, and the experienced difficulty as well as the occurrence of end-of-life discussions. Standard item analyses and an exploratory factor analysis were conducted. Construct validity was analysed through associations between the DEOLD-FI and the aforementioned measures., Results: Questionnaires were obtained from 112 participants (53% response rate; male 54%, mean age 64.9 years [range 33-94]). In the final 23-item version two factors were extracted: 'emotional burden due to end-of-life discussions' (α = 0.90) and "negative attitudes towards end-of-life discussions" (α = 0.91) explaining 69% of the variance (total scale α = 0.93). Construct validity was supported by its significant correlations with the reported difficulty in end-of-life discussions (r = 0.42) and avoidance of cancer communication (r = 0.40 to r = 0.46) and insignificant correlations with quality of life (r = -0.11), distress (r = 0.16), and physical well-being (r = 0.02). Those who had already engaged in end-of-life discussions showed significantly fewer communication barriers., Conclusion: Results provide evidence that the DEOLD-FI is a valid and reliable instrument for the assessment of difficulties in end-of-life discussions. Benefits for clinical practice and research are discussed., (Copyright © 2021 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

The loss of a loved one can have serious health implications. In Germany, however, bereavement care services often provide support regardless of risk or need. A structural framework within which these services are provided systematically and which enables the establishment of qualitative standards throughout Germany has not yet been proposed. A British stepped care model for professionalized bereavement support is actually being discussed internationally. In this paper the British model is adapted to the German context in order to improve nationwide bereavement care services., (Copyright © 2021. Published by Elsevier GmbH.)

Context: Children with life-limiting diseases suffer from gastrointestinal (GI) symptoms. Since the introduction of specialized palliative home care (SPHC) in Germany, it is possible to care for these children at home. In phase 1 of care the aim is to stabilize the patient. In phase 2, terminal support is provided. Objectives: Analysis were performed of the differences between these phases. The causes and modalities/outcome of treatment were evaluated. Methods: A retrospective study was performed from 2014 to 2020. All home visits were analyzed with regard to the abovementioned symptoms, their causes, treatment and results. Results: In total, 149 children were included (45.9% female, mean age 8.17 ± 7.67 years), and 126 patients were evaluated. GI symptoms were common in both phases. Vomiting was more common in phase 2 (59.3 vs. 27.1%; p < 0.001). After therapy, the proportion of asymptomatic children in phase 1 increased from 40.1 to 75.7%; ( p < 0.001 ). Constipation was present in 52.3% (phase 1) and 54.1% (phase 2). After treatment, the proportion of asymptomatic patients increased from 47.3 to 75.7% in phase 1 ( p < 0.001), and grade 3 constipation was reduced from 33.9 to 15% in phase 2 ( p < 0.05). Conclusion: Painful GI symptoms occur in both palliative care phases but are more common in phase 2. The severity and frequency can usually be controlled at home. The study limitations were the retrospective design and small number of patients, but the study had a representative population, good data quality and a unique perspective on the reality of outpatient pediatric palliative care in Germany., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hauch, Kriwy, Hahn, Dettmeyer, Zimmer, Neubauer, Brill, Vaillant, de Laffolie, Schaefer, Tretiakowa, Hach, Sibelius and Berthold.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Pain is a common symptom leading to referrals to specialized home palliative care (SHPC) services and is known to affect patients' quality of life. To date, little is known about the impact of referral source on its management. To assess changes to pain medication profile in the course of SHPC and to identify potential differences in relation to referral source. This exploratory study is a retrospective analysis of 501 electronic medical records of a SHPC team in Germany. This included the assessment of baseline pain medication profiles according to the WHO analgesic ladder and changes to analgesic treatment in the course of SHPC with respect to referral source. At the time of admission, 77.4% of patients referred by a hospital and 78.8% of patients referred by the outpatient sector received a fixed analgesic regimen. In all, 61.9% of the inpatient group versus 62.9% of the outpatient group were treated with opioids, and 79.0% received modifications to pain medication at one point in time following admission. Thereby, patients referred by the outpatient sector received significantly earlier modifications and more supplementations of pain medication. Our study suggests positive development in the prescription of opioid analgesics compared to earlier studies in Germany. On the one hand, it highlights the relevance of thorough assessment and responsive evaluation of pain in SHPC, and on the other hand it reveals possible training needs of referring physicians, particularly those working in the outpatient sector. Our results inspired further research examining more closely the links between referral source and pain management.

Objective: To give an overview of English and German speaking bereavement measurements for difficulties in the grieving process and complicated grief., Method: Systematic literature review., Results: 22 measures were identified. They are categorised into general, specialized and pre-death measurements. All scales proved to have predominantly good psychometric properties., Discussion: Dozens of measures of grieving have been proposed internationally. Only 3 general and 3 specialized scales are available in German language., Conclusion: Up to date no scale exists in German language to assess bereavement difficulties prior to death., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an active TLR-4 pathway. Proteome profiling array revealed that LPS combined with irradiation resulted in a strong phosphorylation of cAMP response element-binding protein (CREB). Inhibition of CREB binding protein (CBP) by the specific inhibitor ICG-001 not only abrogated the LPS-induced radioresistance but even led to an increase in radiosensitivity. The sensitization caused by ICG-001 could be attributed to a reduction of DNA double-strand break (DSB) repair. It is shown that in NSCLC cells LPS leads to a CREB dependent radioresistance which is, however, reversible through CBP inhibition by the specific inhibitor ICG-001. These findings indicate that the combined treatment with radiation and CBP inhibition may improve survival of NSCLC patients suffering from pulmonary infections., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

A 28-year-old Syrian refugee presented with right-sided knee pain and progressive deterioration of the general condition over the past months. Laboratory diagnostics revealed severe hypercalcemia due to primary hyperparathyroidism, and computed tomography (CT) scanning demonstrated disseminated osteolytic lesions throughout the skeleton. Histologically, these lesions were characterized by multinuclear giant cells (defining these lesions as so-called brown tumors). Finally, surgical removal of a jugular mass allowed the histopathologic diagnosis of a sporadic parathyroid carcinoma. In the patient, this condition was associated with a mutation in the HPRT2 gene locus.

Pulmonary infections are frequent complications in lung cancer and may worsen its outcome and survival. Inflammatory mediators are suspected to promote tumor growth in non-small-cell lung cancer (NSCLC). Hence, bacterial pathogens may affect lung cancer growth by activation of inflammatory signalling. Against this background, we investigated the effect of purified lipoteichoic acids (LTA) of Staphylococcus aureus (S. aureus) on cellular proliferation and liberation of interleukin (IL)-8 in the NSCLC cell lines A549 and H226. A549 as well as H226 cells constitutively expressed TLR-2 mRNA. Even in low concentrations, LTA induced a prominent increase in cellular proliferation of A549 cells as quantified by automatic cell counting. In parallel, metabolic activity of A549 cells was enhanced. The increase in proliferation was accompanied by an increase in IL-8 mRNA expression and a dose- and time-dependent release of IL-8. Cellular proliferation as well as the release of IL-8 was dependent on specific ligation of TLR-2. Interestingly, targeting IL-8 by neutralizing antibodies completely abolished the LTA-induced proliferation of A549 cells. The pro-proliferative effect of LTA could also be reproduced in the squamous NSCLC cell line H226. In summary, LTA of S. aureus induced proliferation of NSCLC cell lines of adeno- and squamous cell carcinoma origin. Ligation of TLR-2 followed by auto- or paracrine signalling by endogenously synthesized IL-8 is centrally involved in LTA-induced tumor cell proliferation. Therefore, pulmonary infections may exert a direct pro-proliferative effect on lung cancer growth.

Background: Since the amendment of the Social Law V in Germany in 2007 the financial basis for a Specialised Home Palliative Care for Children (SHPC) for children was established. In Hesse 3 different SHPC teams entered into collective negotiations with health insurance companies. In 2014, the team of the University Children's Hospital in Giessen started to treat the first patient with a lead time of two months., Methods: Thus in this paper the development of a SHPC team is described. After the first year anonymized patients data were retrospectively analyzed., Results: Within 12 months 35 patients, 24 females and 11 males, were treated. All of the 6 patients who died, died at home. Calculated 48 weeks survival was 78%. 45% of the patients suffered from malignancies, 34% of malformations and 34% had metabolic disorders. 51% needed crisis intervention and 51% infusion therapy. Only 26% of parents denied cardiopulmonary resuscitation (CPR). Only 10% of the patients or their families received professional psychological care., Conclusion: Formation of a SHPC is feasible within a short time period once a financial basis is established. So, empathic guidance of families to help decision making for emergency situations are considered to be important. Analysis of patient's data after one year could help to improve the quality of care. Our data provides information for developing a palliative care team und could motivate colleagues to start the job., (© Georg Thieme Verlag KG Stuttgart · New York.)

The inflammatory tumor microenvironment plays a crucial role in tumor progression. In lung cancer, both bacterial infections and neutrophilia are associated with a poor prognosis. In this study, we characterized the effect of isolated human neutrophils on proliferation of the non-small cell lung cancer (NSCLC) cell line A549 and analyzed the impact of A549-neutrophil interactions on inflammatory mediator generation in naive and lipopolysaccharide (LPS)-exposed cell cultures. Co-incubation of A549 cells with neutrophils induced proliferation of resting and LPS-exposed A549 cells in a dose-dependent manner. In transwell-experiments, this effect was demonstrated to depend on direct cell-to-cell contact. This pro-proliferative effect of neutrophils on A549 cells could be attenuated by inhibition of neutrophil elastase activity, but not by oxygen radical neutralization. Correspondingly, neutrophil elastase secretion, but not respiratory burst, was specifically enhanced in co-cultures of A549 cells and neutrophils. Moreover, interference with COX-2 activity by indomethacin or the specific COX-2 inhibitor NS-398 also blunted the increased A549 proliferation in the presence of neutrophils. In parallel, a massive amplification of COX-2-dependent prostaglandin E2 synthesis was detected in A549-neutrophil co-cultures. These findings suggest that direct cell-cell interactions between neutrophils and tumor cells cause release of inflammatory mediators which, in turn, may enhance tumor growth in NSCLC.

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

Anti-neutrophil cytoplasmic antibodies (c-ANCA) targeting proteinase 3 (PR3) are implicated in the pathogenesis of Wegener's granulomatosis (WG). Fulminant disease can present as acute lung injury (ALI). In this study, a model of ALI in WG was developed using isolated rat lungs. Isolated human polymorphonuclear leukocytes (PMNs) were primed with tumour necrosis factor (TNF) to induce surface expression of PR3. Co-perfusion of TNF-primed neutrophils and monoclonal anti-PR3 antibodies induced a massive weight gain in isolated lungs. This effect was not observed when control immunoglobulin G was co-perfused with TNF-primed PMNs. The c-ANCA-induced oedema formation was paralleled by an increase in the capillary filtration coefficient as a marker of increased pulmonary endothelial permeability. In contrast, pulmonary artery pressure was not affected. In the presence of the oxygen radical scavenger superoxide dismutase and a NADPH oxidase inhibitor, c-ANCA-induced lung oedema could be prevented. Inhibition of neutrophil elastase was equally effective in preventing c-ANCA-induced lung injury. In conclusion, anti-PR3 antibodies induced neutrophil mediated, elastase- and oxygen radical-dependent ALI in the isolated lung. This experimental model supports the hypothesis of a pathogenic role for c-ANCA in WG and offers the possibility of the development of therapeutic strategies for the treatment of lung injury in fulminant WG.

Proinflammatory cytokines are centrally involved in tumor progression and survival in non-small cell lung cancer, and both the presence of infiltrating neutrophils and bacterial infection in the lung may indicate a poor prognosis. Against this background, we investigated the effect of the bacterial cell wall component lipopolysaccharide (LPS) on interleukin (IL)-6 and IL-8 synthesis in the non-small cell lung cancer line A549 and in A549-neutrophil cocultures. The LPS induced a dose-dependent and time-dependent release of IL-8 from A549 cells, whereas IL-6 could not be detected. Interestingly, in A549-neutrophil cocultures, IL-8 synthesis was massively amplified and IL-6 was also released, compared with the respective monocultures. The A549 cells were identified as the primary cellular source of these cytokines, as enhanced cytokine mRNA transcription was detected in this cell type, although not in neutrophils in the coculture system. Experiments done in transwells indicated that direct cell-cell contact was a prerequisite for the increased cytokine generation. Inhibition of tumor necrosis factor-alpha bioactivity by neutralizing antibodies and blocking cyclooxygenase-2 activity blunted the enhanced cytokine generation in the coculture system. Amplification of LPS-induced cytokine secretion could be reproduced when the small cell lung cancer cell line H69 was cocultured with neutrophils. When the Gram-positive cell wall component lipoteichoic acid was used instead of LPS, cytokine synthesis was also amplified in A549-neutrophil cocultures, to a similar extent to that observed with LPS. These data indicate that interaction between bacterial pathogens, neutrophils, and tumor cells might amplify the release of proinflammatory cytokines which may promote tumor growth in vivo.

Objective: In sepsis, cardiac function is frequently depressed. Microcirculatory disturbances as evidenced in most organs may extend to the coronary circulation and may play a role in the occurrence of cardiac dysfunction. Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH), pore-forming exotoxins of clinically relevant bacteria, have recently been demonstrated to evoke cardiac dysfunction in isolated rat hearts by activating myocardial eicosanoid metabolism. alpha-Toxin activates synthesis of thromboxane (Tx) A2, ECH of cysteinyl-leukotrienes (Sibelius U, Grandel U, Buerke M, et al: Leukotriene-mediated coronary vasoconstriction and loss in myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused reat hearts. Crit Care Med 2003; 3:683-688, Sibelius U, Grandel U, Buerke M, et al: Staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts-Role of Tx formation. Circulation 2000; 101:78-85). We now investigated whether cardiac dysfunction in response to alpha-toxin and ECH is caused by disturbances of regional cardiac perfusion., Design: A prospective, experimental study., Setting: A research laboratory at a university hospital., Subjects: Isolated hearts from male Wistar rats., Interventions: Changes of regional perfusion were investigated by using colored microspheres in isolated rat hearts perfused with alpha-toxin or ECH either at constant coronary perfusion pressure or constant coronary flow rate. Significance of toxin-activated eicosanoid generation was evaluated by pharmacologic interventions., Measurements and Main Results: By eliciting eicosanoid formation, both toxins caused an increase in coronary vascular resistance and a loss in contractile function. In ECH-perfused hearts, reduction of regional perfusion predominantly occurred in subendocardial sections in either perfusion mode (coronary perfusion pressure or coronary flow rate). When synthesis of cysteinyl-leukotrienes was blocked by the 5-lipooxygenase inhibitor MK-886, disturbances of regional perfusion and the associated cardiac dysfunction were largely prevented. Coronary perfusion of alpha-toxin caused a decrease of regional perfusion that was more pronounced in subepicardial layers. Inhibiting the release of TxA2 by blocking the cyclooxygenase with indomethacin attenuated the perfusion abnormalities and the cardiodepression in response to alpha-toxin., Conclusions: Bacterial exotoxins of clinically relevant bacteria may impair cardiac function by eliciting distinct coronary perfusion abnormalities via release of vasoactive eicosanoids.

Bacterial toxins cause cardiac dysfunction and death through an inflammatory process, but the mechanism remains unclear. Simvastatin is recognized as having anti-inflammatory properties beyond its lipid-lowering effects. We examined Staphylococcus aureus alpha-toxin in isolated heart and in vivo models and tested simvastatin's effects in sepsis. Isolated Langendorff-perfused rat hearts were exposed to a recirculating perfusate containing alpha-toxin (0.5 microg mL(-1)). Compared with controls, there was a significant increase in coronary perfusion pressure and fall in myocardial performance. Significant increases in p53 expression and apoptosis (1.3 +/- 0.5 to 7.1 +/- 1.4 terminal deoxynucleaotidyl transferase nick end labeling-positive cells; P < 0.05) compared with controls were observed, but markers of necrosis were similar. In parallel experiments, anaesthetized rats receiving alpha-toxin (40 microg kg(-1), i.v.) had in vivo hemodynamic parameters and serum markers of necrosis monitored for 4 h before the hearts were analyzed for histological change, p53 expression, and apoptosis. Over 4 h, alpha-toxin exposure produced substantial hemodynamic effects. In addition, p53 expression (0.2 +/- 0.2 to 7.1 +/- 0.5 p53-positive myocytes; P < 0.05), TNF-alpha levels, the degree of apoptosis, and markers of necrosis were all significantly increased compared with control animals. Pretreatment with simvastatin protected against alpha-toxin-induced sepsis associated with reduced p53, TNF-alpha, apoptosis, and necrosis. We found significant changes in systemic hemodynamics, coronary perfusion pressure, myocardial function, and increased p53 expression with apoptosis due to bacterial exotoxin. In vivo changes were significantly inhibited by pretreatment with simvastatin. We provide novel evidence for the mechanisms by which septicemia causes myocardial depression and hint at a potential role for simvastatin as an inhibitor of apoptosis in sepsis.

Objective: In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils., Subjects: Isolated human neutrophils from healthy volunteers., Interventions: Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies., Measurements: Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction., Conclusions: The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Transfusion-related acute lung injury (TRALI) is a hazardous complication of transfusion and has become the leading cause of transfusion-related death in the United States and United Kingdom. Although leukoagglutinating antibodies have been frequently shown to be associated with the syndrome, the mechanism by which they induce TRALI is poorly understood. Therefore, we reproduced TRALI in an ex vivo rat lung model. Our data demonstrate that TRALI induction by antileukocyte antibodies is dependent on the density of the cognate antigen but does not necessarily require leukoagglutinating properties of the antibody or the presence of complement proteins. Rather, antibody-mediated activation of neutrophils seems to initiate TRALI, a process that could be triggered by neutrophil stimulation with fMLP. Antibody-mediated neutrophil activation and subsequent release of reactive oxygen species may thus represent key events in the pathophysiologic cascade that leads to immune TRALI.

Objective: Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH) evoke cardiac dysfunction in isolated rat hearts by provoking myocardial synthesis of arachidonic acid-derived thromboxane A2 or the cysteinyl-leukotrienes, LTC4, LTD4, and LTE4, respectively. We investigated whether low doses of either toxin, which fail to induce cardiac depression by themselves, induce cardiac dysfunction when combined with free arachidonic acid., Design: Prospective, experimental study., Setting: Research laboratory at a university hospital., Subjects: Isolated hearts from male Wistar rats., Interventions: Hearts were perfused with low doses of ECH or alpha-toxin in the absence or presence of arachidonic acid or the alternative eicosanoid precursor eicosapentaenoic acid (EPA)., Measurements and Main Results: Application of low-dose ECH with arachidonic acid increased coronary perfusion pressure, depressed left ventricular contractile function, provoked electrical instability, and induced a release of creatine kinase concomitant with the liberation of LTC4, LTD4, and LTE4 into the perfusate. All events were abolished when formation of cysteinyl-leukotrienes was blocked by the 5-lipoxygenase activity inhibitor MK-886, targeting 5-lipoxygenase activating protein. In the presence of arachidonic acid, low doses of alpha-toxin caused an increase in cerebral perfusion pressure and a decline of contractile performance, attributable to the release of thromboxane A2, as both events were mitigated by the cyclooxygenase-inhibitor indomethacin. High doses of ECH caused cardiac dysfunction even in the absence of arachidonic acid. However, in the presence of EPA, the cardiodepressant effect of ECH was blunted. Release of EPA-derived LTE5 at the expense of arachidonic acid-derived LTC4, LTD4, and LTE4 was noted in these hearts., Conclusions: The potency of the bacterial exotoxins ECH and alpha-toxin to cause coronary vasoconstriction and myocardial depression is dependent on the availability of free arachidonic acid and may be influenced by supplying omega-3 fatty acids as alternative lipid precursors.

In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.

Background: Lipoteichoic acid (LTA) represents a major virulence factor in gram-positive sepsis., Methods and Results: In the present study we perfused isolated rat hearts for 180 minutes with highly purified LTA from Staphylococcus aureus. A progressive decline of left ventricular contractile function paralleled by the expression of myocardial tumor necrosis factor-alpha (TNF-alpha) mRNA and protein as well as the release of TNF-alpha into the perfusate was observed in LTA-perfused hearts. Employment of an anti-TNF-alpha antibody completely prevented the loss in contractile function. When CD14, a prominent pathogen recognition receptor, was blocked by a specific antibody, induction of TNF-alpha mRNA and protein release as well as the associated cardiodepression was diminished in response to LTA. Synthesis of TNF-alpha protein was located to interstitial cells of LTA-challenged hearts as detected by immunohistochemistry. Besides progressive cardiodepression, coronary perfusion pressure (CPP) was moderately increased in LTA-perfused hearts. This was accompanied by the release of thromboxane A2 (TXA2) into the perfusate and the induction of cyclooxygenase (Cox)-2 mRNA and protein in the myocardium. Blocking of TXA2 by the nonspecific Cox inhibitor indomethacin, the thromboxane receptor antagonist daltroban, or the selective Cox-2 inhibitor NS-398 prevented the increase in CPP., Conclusions: LTA causes cardiac depression by activating myocardial TNF-alpha synthesis via CD14 and induces coronary vascular disturbances by activating Cox-2-dependent TXA2 synthesis. These phenomena may contribute to cardiac depression in gram-positive sepsis.

The NB1 glycoprotein (CD177, HNA-2a antigen) is exclusively expressed on human neutrophils. As the clinical significance of CD177 expression is unknown, we investigated its expression in healthy individuals before and after stimulation with granulocyte colony-stimulating factor (G-CSF), in patients with rheumatoid arthritis, viral hepatitis, severe bacterial infections and polycythaemia vera. Expression was quantitatively determined by flow cytometry and by real time polymerase chain reaction. Only G-CSF-stimulated individuals and patients with severe bacterial infections and polycythaemia showed a significantly (P < 0.001) increased CD177 expression compared with healthy individuals, indicating that neutrophil CD177 expression can increase significantly in certain clinical conditions.

Polymorphonuclear neutrophils (PMN) have been implicated in various vascular inflammatory processes. We isolated PMN from venous blood samples of 10 patients with severe primary pulmonary arterial hypertension (PPH), 7 patients with pulmonary hypertension secondary to chronic thromboembolism (CTEPH), and 12 healthy controls. When stimulated with the calcium-ionophore A23187, platelet activating factor (PAF) or the microbial agent n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP), significantly increased release of elastase and superoxide anion was noted in both groups with pulmonary hypertension. Moreover, the neutrophils of CTEPH patients responded with an enhanced liberation of leukotriene (LT) B(4) and 5-hydroxyeicosatetraenoic acid (5-HETE). Inhalation of aerosolized iloprost (5 microg) caused a rapid decline in pulmonary vascular resistance, in both PPH and CTEPH. This hemodynamic response was paralleled by a significant suppression of ionophore- and ligand-induced elastase and superoxide release, as well as LTB(4) and 5-HETE formation. The neutrophil inhibitory effect of the inhalation maneuver was fully reproduced by in vitro incubation of neutrophils with 1-10 pg/ml iloprost for 2 hours. This is the first study to demonstrate that circulating neutrophils from patients with PPH and CTEPH possess an enhanced readiness to respond with inflammatory mediator generation to different stimulatory agents ex-vivo, and that PMN respiratory burst, elastase secretion and leukotriene generation are promptly reduced by an iloprost inhalation maneuver. Neutrophils might participate in the inflammatory processes in pulmonary arterial hypertension.

Objective: To compare the effects of a conventional omega-6 lipid infusion and a fish oil based (omega-3) lipid infusion for parenteral nutrition on neutrophil function, lipid mediators, and plasma free fatty acids., Design and Setting: Open-label, randomized, pilot study in a university hospital medical intensive care unit and experimental laboratory., Patients and Participants: Ten patients with septic shock and eight healthy controls., Interventions: Patients (five per group) requiring parenteral nutrition received intravenously either a omega-3 or a omega-6 lipid emulsion for a 10-day period., Measurements and Results: At baseline levels of plasma free fatty acids were elevated several-fold, including high concentrations of the omega-6 lipid precursor arachidonic acid (AA). Neutrophils isolated from septic patients displayed markedly reduced responsiveness to ex vivo stimulation, including lipid mediator generation [leukotrienes (LT), PAF], respiratory burst, and phosphoinositide hydrolysis signaling. Under the omega-6 lipid infusion regimen abnormalities in plasma free fatty acids and impairment of neutrophil functions persisted or worsened. In contrast, a rapid switch in the plasma free fatty acid fraction to predominance of the omega-3 acids eicosapentaenoic acid and docosahexaenoic acid over AA occurred in response to omega-3 lipid infusion. LTB(5), in addition to LTB(4), appeared upon neutrophil stimulation originating from these patients, and neutrophil function was significantly improved in the omega-3 lipid group., Conclusions: omega-3 vs. omega-6 lipid emulsions differentially influence the plasma free fatty acid profile with impact on neutrophil functions. Lipid-based parenteral nutrition in septic patients may thus exert profound influence on sequelae and status of immunocompetence and inflammation.

Objective: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis. We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow., Design: Prospective, experimental study., Setting: Research laboratory at a university hospital., Subjects: Isolated hearts from male Wistar rats., Interventions: Isolated hearts were perfused with purified hemolysin for 60 min., Measurements and Main Results: Low concentrations of the toxin in the perfusate (0.1-0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase). Moreover, 0.2 hemolytic units/mL hemolysin evoked ventricular fibrillation within 10 mins of toxin application. These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate. The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase. In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective. In addition, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886., Conclusions: Low doses of hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature. Bacterial exotoxins such as hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.

Background: Simvastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, has been shown to lower serum cholesterol levels in clinical use. Moreover, statins exert beneficial effects in vascular diseases by inhibition of leukocyte rolling, adherence, and transmigration. The aim of this study was to determine if pretreatment with simvastatin attenuates Staphylococcus aureus alpha-toxin-induced increase in leukocyte-endothelial interactions during exotoxemia., Methods and Results: The effects of simvastatin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation. Simvastatin (50 or 100 microg/kg) was administered 18 hours before the study. Activation of microcirculation was induced by bolus administration of 40 microg/kg S aureus alpha-toxin. Exotoxemia resulted in a significant and time-dependent increase in leukocyte rolling, adherence, and transmigration of leukocytes as well as P-selectin expression on the intestinal vascular endothelium. Pretreatment with simvastatin significantly inhibited exotoxin-induced leukocyte rolling from 71+/-10 to 14+/-4.7 cells/min (P<0.01) and adherence from 14+/-3.5 to 0.4+/-0.2 cells (P<0.01). In addition, simvastatin pretreatment significantly inhibited transmigration of leukocytes from 10.5+/-1.2 to 4.2+/-0.9 (P<0.05) cells. Immunohistochemical detection of endothelial cell adhesion molecule P-selectin showed a 50% decrease in endothelial cell surface expression after simvastatin treatment. Furthermore, simvastatin treatment resulted in enhanced expression of endothelial cell NO synthase III in the intestinal microcirculation., Conclusions: These results demonstrate that simvastatin interferes with exotoxin-induced leukocyte-endothelial cell interactions, which may be relevant in various infectious diseases. Statin treatment may offer a new therapeutic strategy for these clinical conditions.

Objective: Exposure of neutrophils to low doses of bacterial lipopolysaccharides enhances their readiness to respond with inflammatory mediator generation including oxygen radical formation to a subsequently applied inflammatory stimulus ("priming"). In the present study, we investigated the role of lipid mediator synthesis and the impact of the anti-inflammatory cytokine interleukin-10 on the lipopolysaccharide-dependent priming of human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine., Design: Prospective, experimental study., Setting: Research laboratory at a university hospital., Subjects: Isolated neutrophils from healthy volunteers., Interventions: Incubation of isolated neutrophils with endotoxin., Measurements and Main Results: Evidence for two distinct priming mechanisms was obtained. The first was strictly serum component dependent, proceeded via CD14, and was not inhibited by even high concentrations of interleukin-10. The second priming mechanism was serum component independent but nevertheless proceeded via CD14. It was linked with neutrophil synthesis of the platelet activating factor and resulted in the appearance of leukotrienes, in particular leukotriene B4, as far as exogenous arachidonic acid was provided. The employment of a platelet-activating factor receptor antagonist (WEB 2086) blocked leukotriene synthesis, and both WEB 2086 and a 5-lipoxygenase inhibitor (MK-886) suppressed the respiratory burst linked with this second priming pathway. This sequence of priming events was inhibited by interleukin-10, when this cytokine was coadministered with the priming agent lipopolysaccharide, whereas late interleukin-10 admixture was ineffective., Conclusions: We conclude that two mechanisms of lipopolysaccharide priming of human neutrophil respiratory burst can be differentiated. One displays serum component dependence, is independent of neutrophil lipid mediator generation, and is not affected by interleukin-10. The other is serum independent although being operated via CD14, employs autocrine loops of platelet-activating factor and leukotriene B4 synthesis, and is sensitive to the inhibitory capacity of interleukin-10. These features may be relevant when the goal is to pharmacologically modify neutrophil functions in septic events.

Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c-ANCA)], a leukocyte serine protease, are highly specific for Wegener's Granulomatosis (WG). A pathogenetic role for c-ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti-PR3 antibodies (anti-PR3) and c-ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti-PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1beta and delayed release of IL-6, IL-8, and thromboxane A2 (TxA2). The secretory response was reproduced by c-ANCA but not by human and murine control IgG and anti-CD14 antibodies. Because F(ab)2 fragments of anti-PR3 were ineffective, coligation of Fc gamma receptors (FcgammaR) was apparently mandatory for monocyte activation. Using soluble receptors for TNF-alpha and IL-1beta and a Tx receptor antagonist, we noted that the "early" cytokines functioned as inducers of TxA2, which then activated IL-8 release. In contrast, IL-6 formation was an independent event. We concluded that anti-PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF-alpha, IL-1beta, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.

Among the anti-neutrophil cytoplasmic Abs (ANCA), those targeting proteinase 3 (PR3) have a high sensitivity and specificity for Wegener's granulomatosis (WG). A pathogenetic role for these autoantibodies has been proposed due to their capacity of activating neutrophils in vitro. Recently, PR3 was also detected in human renal tubular epithelial cells (TEC). In the present study, the effect of murine monoclonal anti-PR3 Abs (anti-PR3) and purified c-ANCA targeting PR3 from WG serum on isolated human renal tubular cell signaling and inflammatory mediator release was characterized. Priming of TEC with TNF-alpha resulted in surface expression of PR3, as quantified in immunofluorescence studies and by flow cytometry. Moreover, PR3 was immunoprecipitated on surface-labeled TEC. Primed TEC responded to anti-PR3 with a dose- and time-dependent activation of phosphoinositide hydrolysis, resulting in a remarkable accumulation of inositolphosphates. Control IgG was entirely ineffective, whereas PR3-ANCA reproduced the phosphoinositide response. The signaling response was accompanied by a pronounced release of superoxidanion into the cell supernatant. Moreover, large amounts of PGE(2) and, to a lesser extent, of thromboxane B(2), the stable metabolite of TxA(2), were secreted from anti-PR3-stimulated TEC. In parallel, a rise in intracellular cAMP levels was observed, which was blocked by the cyclooxygenase inhibitor indomethacin. We conclude that anti-PR3 Abs directly target renal TECs, thereby provoking pronounced activation of the phosphoinositide-related signal transduction pathway. Associated metabolic events such as the release of reactive oxygen species and lipid mediators may directly contribute to the development of renal lesions and loss of kidney function in WG.

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.

The effect of Staphylococcus aureus alpha toxin (alpha-toxin) on selectin-mediated neutrophil adhesion was investigated in polymorphonuclear leukocyte- (PMN) induced vasocontraction and endothelial dysfunction. Adherence of human PMNs to rat aortic endothelium increased significantly following stimulation of the endothelium with alpha-toxin (0.1, 0.5, and 1 microg/mL). This effect could be significantly attenuated by monoclonal antibodies directed against P-selectin or fucoidin, a carbohydrate known to block selectins. Unstimulated human PMNs (10(6)cells/mL) were added to organ chambers containing rat aortic rings stimulated with alpha-toxin (0.5 microg/mL). PMNs elicited a significant vasocontraction in alpha-toxin-stimulated, but not in control aortic, rings (142+/-12 mg versus 12+/-4 mg, P < 0.05). This PMN-induced vasocontraction was virtually blunted by pretreatment with MAb directed against P-selectin or fucoidin (P < 0.05). Endothelial function as assessed by endothelium-dependent vasorelaxation to acetylcholine was substantially inhibited after induction of PMN-induced vasocontraction in alpha-toxin-stimulated aortic rings. This endothelial dysfunction was reduced by P-selectin MAb or fucoidin. In contrast, endothelium-independent relaxation to sodium nitrite was not altered by PMN incubation, indicating that vascular smooth muscle function was unaffected. Thus, PMN-endothelial interaction following S. aureus a-toxin activation of the vascular endothelium is at least, in part, mediated by selectins. As a consequence, PMN-induced vasocontraction and endothelial dysfunction occur. Such mechanisms may be involved in microcirculation abnormalities encountered in sepsis or septic shock due to S. aureus infection.

Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 x 10(6) PMN/ml; 60 x 10(6) PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-alpha and IL-1beta rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking beta(2) integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 x 10(6) PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1beta- and TNF-alpha-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.

The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca(2+) suggested LLO-elicited Ca(2+) flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcA mutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-kappa B to the stimulation with the LLO-producing strains EGD and Listeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-kappa B activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-kappa B translocation and the upregulation of cytokine synthesis. We conclude that L. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca(2+) flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-kappa B are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.

Objective: Myocardial depression, which frequently occurs in the course of septic shock, has been attributed to the cardiodepressant properties of nitric oxide (NO) generated by either the inducible NO synthase (iNOS) or the constitutive isoform (cNOS). We have previously demonstrated that alpha-toxin from Staphylococcus aureus induces thromboxane-mediated vasoconstriction accompanied by severe cardiodepression in isolated rat hearts. In the present study, we investigated the role of NO in the alpha-toxin-induced vascular and contractile abnormalities., Design: Prospective, experimental study., Setting: Research laboratory at a university hospital., Subjects: Isolated hearts from male Wistar rats., Interventions: Isolated hearts were perfused with purified staphylococcal alpha-toxin for 60 mins., Measurements and Main Results: At a concentration of 0.25 and 0.5 microg/mL, alpha-toxin induced a rise in coronary perfusion pressure, depressed myocardial contractility, and caused edema formation. Simultaneously, a time- and dose-dependent rapid release of NO into the perfusate was noted as quantified by a chemiluminescence technique. L-NMMA, a nonselective inhibitor of NOS, but not PBITU, an iNOS-selective inhibitor, blocked NO synthesis, markedly increased the rise in coronary perfusion pressure and the loss in contractility, and enhanced edema formation in response to alpha-toxin. In contrast, zaprinast, a selective inhibitor of phosphodiesterase type V that is used for stabilization of cyclic guanosine monophosphate, attenuated the toxin-induced coronary vasoconstrictor response and the myocardial depression. L-arginine, the substrate of NOS, had similar, yet less potent, effects as zaprinast and slightly increased the release of NO caused by alpha-toxin. Immunohistochemical analysis of the myocardium at the end of the perfusion period demonstrated a positive staining for cNOS but not for iNOS. In addition, no up-regulation of iNOS mRNA was detected in the tissue of toxin-exposed hearts., Conclusions: Staphylococcal alpha-toxin provokes NO biosynthesis via activation of cNOS in rat hearts. NO partly antagonizes the deleterious effects of this pathogenicity factor on coronary vasoregulation and myocardial performance.

Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 microg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5-7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis.

Background: Although endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated., Methods and Results: Perfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-alpha into the perfusate and preceded by myocardial TNF-alpha mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-alpha antiserum or the ceramidase inhibitor N:-oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A(2) and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-alpha and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban., Conclusions: LPS depresses contractility of isolated rat hearts by inducing TNF-alpha synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2-derived TxA(2) appears to facilitate TNF-alpha synthesis in response to LPS.

Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. Free oxygen radicals have been implicated in the pathogenesis of cardiovascular disorders. Therefore, we aimed to test the hypothesis that increased oxidative stress constitutes one underlying mechanism for the connection between OSA and cardiovascular disease. In 18 patients with OSA the release of superoxide from polymorphonuclear neutrophils was determined after stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine (fMLP) and the calcium ionophore A23. Superoxide production was measured as superoxide dismutase-inhibitable reduction of cytochrome c. Blood samples were obtained before and after two nights of CPAP therapy and after 4.8 +/- 0.6 mo of follow-up. Ten healthy young volunteers and 10 lung cancer patients without OSA but a similar spectrum of comorbidity served as controls. Before CPAP, neutrophil superoxide generation was markedly enhanced in OSA when compared with both control groups. Effective CPAP therapy led to a rapid and long-lasting decrease of superoxide release in OSA. In conclusion, OSA is linked with a "priming" of neutrophils for enhanced respiratory burst. The increased superoxide generation, which might have major impact on the development of cardiovascular disorders, is virtually fully reversed by effective CPAP therapy.