Therapeutic targeting of endoplasmic reticulum stress in acute graft-versus-host disease.

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), a potentially curative treatment for leukemia. Endoplasmic reticulum (ER) stress occurs when the protein folding capacity of the ER is oversaturated. How ER stress modulates tissue homeostasis in the context of alloimmunity is not well understood. We show that ER stress contributes to intestinal tissue injury during GvHD and can be targeted pharmacologically. We observed high levels of ER stress upon GvHD onset in a murine allo- HCT model and in human biopsies. These levels correlated with GvHD severity, underscoring a novel therapeutic potential. Elevated ER stress resulted in increased cell death of intestinal organoids. In a conditional knockout model, deletion of the ER stress regulator transcription factor Xbp1 in intestinal epithelial cells induced a general ER stress signaling disruption and aggravated GvHD lethality. This phenotype was mediated by changes in the production of antimicrobial peptides and the microbiome composition as well as activation of pro-apoptotic signaling. Inhibition of inositol-requiring enzyme 1α (IRE1α), the most conserved signaling branch in ER stress, reduced GvHD development in mice. IRE1α blockade by the small molecule inhibitor 4m8c improved intestinal cell viability, without impairing hematopoietic regeneration and T-cell activity against tumor cells. Our findings in patient samples and mice indicate that excessive ER stress propagates tissue injury during GvHD. Reducing ER stress could improve the outcome of patients suffering from GvHD.

Bile acids regulate intestinal antigen presentation and reduce graft-versus-host disease without impairing the graft-versus-leukemia effect.

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute graft-versus-host disease. Here, we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid, the most potent compound in our in vitro studies, reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation.

Immunomodulatory effects of preparations from Anthroposophical Medicine for parenteral use.

BACKGROUND: Preparations from anthroposophical medicine (AM) are clinically used to treat inflammatory disorders. We wanted to investigate effects of a selection of AM medications for parenteral use in cell-based systems in vitro. METHODS: Colchicum officinale tuber D3, Mandragora D3, Rosmarinus officinale 5% and Bryophyllum 5% were selected for the experiments. Induction of apoptosis and necrosis (human lymphocytes and dendritic cells [DCs]) and proliferation of lymphocytes as well as maturation (expression of CD14, CD83 and CD86) and cytokine secretion (IL-10, IL12p70) of DCs were analyzed. Furthermore, proliferation of allogeneic human T lymphocytes was investigated in vitro in coculture experiments using mature DCs in comparison to controls. RESULTS: The respective preparations did not induce apoptosis or necrosis in lymphocytes or DCs. Lymphocyte proliferation was dose-dependently reduced by Colchicum officinale tuber D3 while the viability was unchanged. Rosmarinus officinale 5%, but not the other preparations, dose-dependently inhibited the maturation of immature DCs, reduced secretion of IL-10 and IL-12p70 and slightly inhibited proliferation of allogeneic CD4(+) T-lymphocytes in coculture experiments with DCs. CONCLUSION: The selected preparations from AM for parenteral use are nontoxic to lymphocytes and DCs. Rosmarinus officinale 5% has immunosuppressive properties on key functions of the immune system which propose further investigation.

Equisetum arvense (common horsetail) modulates the function of inflammatory immunocompetent cells.

BACKGROUND: In Europe, extracts of Equisetum arvense (common horsetail) have a long tradition in the treatment of inflammatory disorders. To understand the molecular basis for its use, we investigated the immunomodulatory capacity of a standardized commercially available common horsetail extract on human primary lymphocyte function in vitro. METHODS: The standardized extract of Equisetum arvense was phytochemically characterized. Effects on proliferation, viability and activity of mitogen-activated human lymphocytes were assessed in comparison to cyclosporine A using annexin V/propidium iodide staining assays and flow cytometry-based surface receptor characterization, respectively. Intracellular levels of effector molecules (IL-2, IFN-γ and TNF-α) were analyzed with cytokine assays. RESULTS: T cell proliferation was inhibited dose dependently by the Equisetum extract without induction of apoptosis or necrosis. This effect was mediated through inhibition of lymphocyte activation, specifically by diminishing CD69 and IL-2 surface receptor expression and intracellular IL-2 production. Furthermore, treatment with Equisetum arvense inhibited effector functions, as indicated by reduced production of IFN-γ and TNF-α. CONCLUSIONS: The data indicate that the used extract of Equisetum arvense interferes with the polyfunctionality of immunocompetent cells thereby providing an anti-inflammatory mode-of-action.

"Scanxiety" and a sense of control: the perspective of lung cancer survivors and their caregivers on follow-up - a qualitative study.

OBJECTIVES: Lung cancer survivors often suffer from physical, emotional and social long-term effects of disease and treatment. Caregivers are also affected by the cancer diagnosis throughout the course of the disease and are frequently burdened by high levels of psychosocial stress. However, little is known about how follow-up care after the completed treatment phase can help to improve long-term quality of life. In the context of patient-centred cancer care, considering the survivors' and caregivers' perspectives is an important step toward improving care structures. We therefore explored how lung cancer survivors and their caregivers experience follow-up examinations and their possible psychosocial effects on everyday life in order to shed light on what support is helpful for improving their quality of life. MATERIAL AND METHODS: 25 survivors after curative lung cancer treatment and 17 caregivers underwent a face-to-face semi-structured, audio-recorded interview that was analysed using qualitative content analysis. RESULTS: Especially burdened cancer survivors and caregivers described recurring anxiety before a follow-up appointment influencing their everyday life. At the same time, follow-up care also provided reassurance of still being healthy and helped regain a sense of security and control until the following scan. Despite possible long-term consequences in everyday life, the interviewees reported that the survivors´ psychosocial needs were not explicitly assessed or discussed. Nevertheless, the interviewees indicated that conversations with the physician were important for the success of "good" follow-up care. CONCLUSION: Anxiety surrounding follow-up scans, also known as "scanxiety", is a common problem. In this study, we expanded on previous findings and found a positive aspect of scans, namely regaining a sense of security and control, which can strengthen the psychological well-being of the survivors and their families. To optimize follow-up care and improve the quality of life of lung cancer survivors and caregivers, strategies to integrate psychosocial care, like the introduction of survivorship care plans or increased use of patient-reported outcomes, should be explored in the future.

Metabolic reprogramming of donor T cells enhances graft-versus-leukemia effects in mice and humans.

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post-allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.

IFN-y enhances T(H)1 polarisation of monocyte-derived dendritic cells matured with clinical-grade cytokines using serum-free conditions.

Using serum-free conditions, human monocyte-derived dendritic cells (MoDCs) tend to mature insufficiently in a T(H)1-polarizing direction under approved and standardized clinical conditions. However, for the initiation of an efficient tumour antigen-specific cytotoxic T-cell response, the induction of a distinct T(H)1 response is favourable. Therefore, to improve T(H)1 polarisation, the influence of interferon-gamma (IFN-gamma) on the maturation of MoDCs was investigated with clinical-grade cytokines or lipopolysaccharide (LPS) in serum-free medium focusing on the viability, phenotypic characteristics, cytokine profile and restimulating capacities. As in previous research, we confirmed that in respect of viability and phenotypic characteristics, cytokine cocktails consisting of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and prostaglandin (PG) E2, mature MoDCs most efficiently. However, these cytokine-matured MoDCs secreted relatively high levels of IL-10 and only low levels of IL-12p70. Remarkably, if IFN-gamma was added, significantly lower levels of IL-10 concomitant with higher levels of IL-12p70 could be detected. Pretreatment with IFN-gamma did not improve the phenotypic characteristics nor the T(H)1 polarisation of MoDCs. Nevertheless, MoDCs matured with clinical-grade cytokines and IFN-gamma could be re-stimulated most effectively with IFN-gamma. In conclusion, our work demonstrates that addition of INF-gamma to clinical-grade cytokine cocktails readily matures MoDCs and enhances their T(H)1 polarisation efficiently under serum-free conditions.

Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model.

Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies.

Immunosuppressive activity of an aqueous Viola tricolor herbal extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Heartsease (Viola tricolor L.), a member of the Violaceae family, has a long history as a medicinal plant and has been documented in the Pharmacopoeia of Europe. Due to its anti-inflammatory properties it is regarded as a traditional remedy against skin diseases, for example for the treatment of scabs, itching, ulcers, eczema or psoriasis, and it is also used in the treatment of inflammation of the lungs and chest such as bronchitis or asthma. Because T-cells play an important role in the pathological process of inflammatory diseases we investigated the effect of an aqueous Viola extract on lymphocyte functions and explored the 'active' principle of the extract using bioactivity-guided fractionation. MATERIAL AND METHODS: An aqueous Viola extract was prepared by C18 solid-phase extraction. Effects on proliferation of activated lymphocytes (using the cell membrane permeable fluorescein dye CFSE), apoptosis and necrosis (using annexin V and propidium iodide staining), interleukin-2 (IL-2) receptor expression (using fluorochrome-conjugated antibodies) and IL-2 cytokine secretion (using an ELISA-based bead array system) were measured by flow cytometry. Influence on lymphocyte polyfunctionality was characterized by Viola extract-induced production of IFN-γ and TNF-α, as well as its influence on lymphocyte degranulation activity. Fractionation and phytochemical analysis of the extract were performed by RP-HPLC and mass spectrometry. RESULTS: The aqueous Viola extract inhibited proliferation of activated lymphocytes by reducing IL-2 cytokine secretion without affecting IL-2 receptor expression. Similarly, effector functions were affected as indicated by the reduction of IFN-γ and TNF-α production; degranulation capacity of activated lymphocytes remained unaffected. Bioassay-guided fractionation and phytochemical analysis of the extract led to identification of circular plant peptides, so called cyclotides, as bioactive components. CONCLUSION: An aqueous Viola extract contains bioactive cyclotides, which inhibit proliferation of activated lymphocytes in an IL-2 dependent manner. The findings provide a rationale for use of herbal Viola preparations in the therapy of disorders related to an overactive immune system. However, further studies to evaluate its clinical potency and potential risks have to be performed.

Traditionally used Veronica officinalis inhibits proinflammatory mediators via the NF-κB signalling pathway in a human lung cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from Veronica officinalis L. are traditionally used for the treatment of lung diseases; however, the effective compounds and the mode of action are still unknown. AIM OF THE STUDY: Here we analyzed the effects of a standardized Veronica extract on genes expression and signalling protein production associated with the development of inflammatory lung diseases. MATERIAL AND METHODS: The degranulation capacity of primary mast cells, as well as gene expression and release of inflammatory mediators from human lung epithelial cells (A549 cells) were analyzed in relation to the synthetic drugs azelastine and dexamethasone. Gene and protein expression of cyclooxygenase-2 were investigated by semi-quantitative RT-PCR and western blotting, respectively. The involvement of phosphorylated mitogen-activated protein kinases and NF-κB signaling in regulation of these molecules were characterized by western blotting and electrophoretic mobility shift assays. Characteristic extract components were identified by LC-MS and verminoside was quantified by HPLC analysis. RESULTS: We demonstrated that Veronica officinalis has a small influence on the degranulation capacity of mast cells but rather inhibits gene and protein expression of the chemokine eotaxin in A549 lung epithelial cells, which is essential for recruitment of inflammatory-associated cells in lung diseases. Furthermore, release of the inflammatory mediator PGE(2) was diminished through inhibition of COX-2 expression via the NF-κB signaling pathway in TNF-α-activated A549 cells. Phytochemical analysis identified verproside and verminoside as the most abundant iridoid glycosides. CONCLUSION: Our results are a contribution to explaining the observed anti-inflammatory effects of Veronica offcinalis extract on a molecular level. However, its clinical potency has at first to be proven in animals and subsequently in clinical trials.

Student-directed learning in a community geriatrics advanced pharmacy practice experience.

OBJECTIVES: To assess a community geriatrics advanced pharmacy practice experience (APPE) that aimed to improve students' attitudes towards older adults and provide a student-directed learning experience. DESIGN: Students provided blood pressure monitoring and medication counseling to older adults living in a low-income residential facility as part of a required 6-week ambulatory care service-learning APPE. Pre-experience and post-experience essays on students' perceptions of the elderly and their intended and actual learning were retrospectively reviewed using a qualitative process to determine whether the course objectives were met. ASSESSMENT: Many students initially described older adults in factual terms or using negative descriptors. Most expressed a desire to increase their knowledge of diseases commonly occurring in and drugs commonly prescribed for the elderly or to improve specific skills. Many students initially had difficulty articulating clear and measurable learning objectives and appropriate assessment metrics, which are important components of self-directed learning. The final essays revealed many students learned more about the humanistic aspects of care than they had anticipated. CONCLUSION: This community-based geriatrics experience improved students' attitudes towards working with older adults and provided practice in developing and assessing their personal learning objectives.

Factors associated with uncontrolled hypertension in an affluent, elderly population.

OBJECTIVE: To explore demographic, economic, and treatment-related associations with uncontrolled hypertension in a relatively affluent, community-dwelling population. METHODS: Two home surveys of 655 and 432 subjects with a diagnosis of hypertension were completed in 1995 and 1999, respectively. Trained surveyors recorded medication history plus demographic and socioeconomic variables, as well as several measurements of blood pressure in the home. Uncontrolled hypertension was defined according to guidelines set forth by the Sixth Report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. Odds ratios for observing an elevated blood pressure given the patients' medication history, income, insurance coverage, demographic variables, and concomitant disease were estimated using multivariable logistic regression. RESULTS: On both occasions, >60% of the study population had an average blood pressure that was above recommended levels. Diuretic therapy alone (1995 survey) or in combination with an angiotensin-converting enzyme inhibitor (1999 survey) decreased the odds ratio for uncontrolled hypertension by 39% (p = 0.027) and 62% (p = 0.005), respectively. Lack of prescription drug insurance and a history of arthritis had negative effects, increasing the odds ratio for uncontrolled hypertension by more than twofold (p < 0.05). CONCLUSIONS: Uncontrolled hypertension occurs commonly among elderly patients notwithstanding a relatively high standard of living. The choice of medication, presence of concomitant disease, and availability of insurance may be important factors influencing blood pressure control.