Junctional adhesion molecule-A deficient mice are protected from severe experimental autoimmune encephalomyelitis.

In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), early pathological features include immune cell infiltration into the central nervous system (CNS) and blood-brain barrier (BBB) disruption. We investigated the role of junctional adhesion molecule-A (JAM-A), a tight junction protein, in active EAE (aEAE) pathogenesis. Our study confirms JAM-A expression at the blood-brain barrier and its luminal redistribution during aEAE. JAM-A deficient (JAM-A-/-) C57BL/6J mice exhibited milder aEAE, unrelated to myelin oligodendrocyte glycoprotein-specific CD4+ T-cell priming. While JAM-A absence influenced macrophage behavior on primary mouse brain microvascular endothelial cells (pMBMECs) under flow in vitro, it did not impact T-cell extravasation across primary mouse brain microvascular endothelial cells. At aEAE onset, we observed reduced lymphocyte and CCR2+ macrophage infiltration into the spinal cord of JAM-A-/- mice compared to control littermates. This correlated with increased CD3+ T-cell accumulation in spinal cord perivascular spaces and brain leptomeninges, suggesting JAM-A absence leads to T-cell trapping in central nervous system border compartments. In summary, JAM-A plays a role in immune cell infiltration and clinical disease progression in aEAE.

[Multifamily Therapy with Anorectic Children and Adolescents in a Day Care Clinic]. / Multifamilientherapie mit anorektischen Kindern und Jugendlichen im tagesklinischen Rahmen.

Multifamily Therapy with Anorectic Children and Adolescents in a Day Care Clinic The treatment of children and adolescents suffering from anorexia nervosa represents an enormous challenge for therapists and families as well. After release form inpatient hospital treatment, there frequently is a period of renewed weight loss and a return to anorectic comportment and thought patterns. In view of what may be understood as a relapse, feelings of guilt and shame on the part of both the patients and their parents can be triggered. This often supports dysfunctional relationship dynamics within the affected families. Maintaining anorexia nervosa may be facilitated by being a suitable strategy to solve problems of the family system. Multifamily therapy aims at breaking this cycle and ensuring a stable transition from inpatient to outpatient settings of therapy. It requires an intensive and continuous involvement of the patients' parents in the therapy. Six families participating in a closed group are accompanied over one year by two multifamily therapists during the post inpatient phase. During this period, the parents remain responsible for their children. In the course of a day hospital intervention taking fifteen single days over a year, the parents develop a sense of competence in dealing with the disease. Thereby, the patients get the opportunity to overcome the anorectic isolation and pseudo-autonomy through experiencing their parents as able to understand them and their illness. In this contribution we present first results of a pilot study that suggest effectiveness of this therapy method.

Limited Ventricular Preload is the Main Reason for Reduced Stress Reserve After Atrial Baffle Repair.

The atrial baffle repair (ABR) significantly improved the fate of patients with transposition of the great arteries (TGA). However, these patients show impaired exercise tolerance and some present severe decline of systemic ventricular function. Intrinsic myocardial weakness, low heart rate response to exercise and diastolic filling impairment are discussed to be causative. Forty-nine long-term survivors with TGA (median age 23.7 year) after ABR were catheterized with measured oxygen consumption in four conditions (baseline, volume, atrial pacing, dobutamine) and the results were compared to 10 normal controls. Median cardiac output was significantly lower in the ABR group (2.2 vs. 2.6 l/min/m2; p = 0.015), and systemic resistance was significantly elevated (28.9 vs. 22.2 U m2; p = 0.04) in comparison with normals. While stroke volume rose by 27% in the control group, it dropped by 7% in patients after ABR at atrial pacing (80/min). Stroke volume increase after dobutamine was significantly lower after ABR in comparison with normal controls (34 vs. 106%; p = 0.001). Higher NYHA class (p = 0.043), degree of tricuspid regurgitation (p = 0.009) and ventricular function (p = 0.028) were associated with lower stroke volume increase. Limited exercise capability of patients after ABR for TGA is primarily due to limited diastolic filling of the ventricles due to stiff non-compliant atrial pathways. Elevated systemic resistance may lead to severe myocardial hypertrophy with possible ischemia and contribute to the multifactorial decline of ventricular function in some patients.

Differentiation and gene expression profile of tumor-associated macrophages.

Tumor microenvironment is composed of proliferating neoplastic cells, a vascular network of endothelial cells, extra cellular matrix produced by fibroblasts, cellular compartments of adaptive immunity like lymphocytes and dendritic cells as well as cells of innate immunity, e.g., natural killer cells and macrophages. Many pre-clinical and clinical studies demonstrate an inversed correlation between macrophage infiltrate and patients' prognosis indicating a macrophage supporting role for tumor progression as producers of growth and angiogenic factors and as regulators of tissue remodelling. Based on in vitro models, macrophages have been classified in pro-inflammatory, classically activated macrophages (M1; stimulated by IFN-γ or LPS) and anti-inflammatory, alternatively activated macrophages (M2; stimulated by either IL-4/IL-13, IL-1ß/LPS in combination with immune complexes or by IL-10/TGFß/glucocorticoids). Tumor escape has been linked with a switch from M1 activation in the early tumor initiation process towards M2-like phenotype during tumor progression, a process that highlights the heterogeneity and plasticity of macrophage activation and which offers a possible therapeutic target directed against reversing the TAM phenotype in the tumor. Here, we review different tumor-environmental stimuli and signalling cascades involved in this switch in differentiation and the so connected gene regulation in TAMs. In addition, therapeutic applications deducted from this differentiation and gene regulatory processes are presented. Data from pre-clinical as well as clinical studies clearly support the notion, that TAMs are excellent novel therapeutic targets for the fight against cancer.

Titanium induces pro-inflammatory and tissue-destructive responses in primary human macrophages.

Implants and medical devices are efficient and practical therapeutic solutions for a multitude of pathologies. Titanium and titanium alloys are used in orthopedics, dentistry, and cardiology. Despite very good mechanical properties, and corrosion resistance titanium implants can fail due to inflammatory or tissue-degradation related complications. Macrophages are major immune cells that control acceptance of failure of the implant. In this study, for the first time, we have performed a systematic analysis of the response of differentially activated human macrophages (M(Control), M(IFNγ) and M(IL-4)) to the polished and porous titanium surfaces in order to identify the detrimental effect of titanium leading to the tissue destruction and chronic inflammation. Transcriptome analysis revealed that the highest number of differences between titanium and control settings are found in M(IL-4) that model healing type of macrophages. RT-qPCR analysis confirmed that both polished and porous titanium affected expression of cytokines, chitinases/chitinase-like proteins and matrix metalloproteinases. Titanium-induced release and activation of MMP7 by macrophages was enhanced by fibroblasts in both juxtacrine and paracrine cell interaction models. Production of titanium-induced MMPs and cytokines associated with chronic inflammation were independent of the presence of Staphylococcus aureus. MMP7, one of the most pronounced tissue-destroying factor and chitinase-like protein YKL-40 were expressed in CD68+ macrophages in peri-implant tissues of patients with orthopedic implants. In summary, we demonstrated that titanium induces pro-inflammatory and tissue-destructing responses mainly in healing macrophages, and the detrimental effects of titanium surfaces on implant-adjacent macrophages are independent on the bacterial contamination.

The CD20 homolog Ms4a8a integrates pro- and anti-inflammatory signals in novel M2-like macrophages and is expressed in parasite infection.

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.

Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, glucocorticoids) and tumor-conditioned media (TCM). Admixture of MS4A8A(+) TCM/IL-4/GC-treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage-like cells displayed a special gene signature. Admixture of these MS4A8A(+) Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF-κB activation and of the MAPK JNK and ERK did not show relevant effects, the p38α/ß MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A(+) TAM may serve as a novel cellular target for selective cancer therapy.

The fusion protein of respiratory syncytial virus triggers p53-dependent apoptosis.

Infection with respiratory syncytial virus (RSV) frequently causes inflammation and obstruction of the small airways, leading to severe pulmonary disease in infants. We show here that the RSV fusion (F) protein, an integral membrane protein of the viral envelope, is a strong elicitor of apoptosis. Inducible expression of F protein in polarized epithelial cells triggered caspase-dependent cell death, resulting in rigorous extrusion of apoptotic cells from the cell monolayer and transient loss of epithelial integrity. A monoclonal antibody directed against F protein inhibited apoptosis and was also effective if administered to A549 lung epithelial cells postinfection. F protein expression in epithelial cells caused phosphorylation of tumor suppressor p53 at serine 15, activation of p53 transcriptional activity, and conformational activation of proapoptotic Bax. Stable expression of dominant-negative p53 or p53 knockdown by RNA interference inhibited the apoptosis of RSV-infected A549 cells. HEp-2 tumor cells with low levels of p53 were not sensitive to RSV-triggered apoptosis. We propose a new model of RSV disease with the F protein as an initiator of epithelial cell shedding, airway obstruction, secondary necrosis, and consequent inflammation. This makes the RSV F protein a key target for the development of effective postinfection therapies.

The novel immunoglobulin super family receptor SLAMF9 identified in TAM of murine and human melanoma influences pro-inflammatory cytokine secretion and migration.

Melanoma is a highly immunogenic tumor with a good response to treatment with immune checkpoint inhibitors. Tumor-associated macrophages (TAMs) play an important immunosuppressive role in such tumors and have therefore been identified as possible future therapeutic targets in oncology. The aim of this study was to identify novel immunoregulatory receptors specifically expressed on TAM. Expression of Slamf9, a member of the signaling lymphocytic-activating molecule (Slam) immunoreceptor family, was found to be upregulated in a gene expression analysis of murine bone marrow-derived macrophages (BMDM) stimulated with tumor-conditioned medium of B16F1 melanoma cells. SLAMF9+ macrophages were identified in human and murine melanomas by using self-generated antibodies against human and murine SLAMF9. A comprehensive immunohistochemical analysis of tissue microarrays detected SLAMF9+ TAM in 73.3% of human melanomas, but also in 95.5% of naevi of melanoma patients and in 50% of naevi from healthy controls. In addition, 20% of melanomas and 2.3% of naevi from melanoma patients displayed a positive SLAMF9 expression also in melanocytic cells. No SLAMF9 expression was detected in naevus cells of healthy donors. Although SLAMF9 has no intracellular signaling motif, a comprehensive functional analysis revealed that the molecule was able to significantly enhance TNF-α secretion after LPS-stimulation. In addition, SLAMF9 delayed the wound closure of RAW 264.7 cells in a scratch assay, while proliferation and cell death were not affected. Taken together, SLAMF9 is a novel type-I-transmembrane receptor with immunomodulatory properties in macrophages. Further studies are required to evaluate whether SLAMF9 classifies as a promising future therapeutic target in melanoma.

IL-4 driven transcription factor FoxQ1 is expressed by monocytes in atopic dermatitis and stimulates monocyte migration.

Monocytes are actively recruited at sites of chronic inflammation. However, molecular factors involved in this process are not fully elucidated. Here, we show that cytokine IL-4 which is implicated in the development of chronic inflammatory disease atopic dermatitis (AD) induces expression of transcription factor FoxQ1 in human monocytes and macrophages. FoxQ1 mRNA levels were elevated in monocytes of AD patients compared to healthy donors. Overexpression of FoxQ1 in RAW 264.7 monocytic cells facilitated their migration towards MCP-1 and was associated with decreased expression of migration-regulating genes (claudin 11 and plexin C1). Furthermore, FoxQ1 overexpression in RAW cells accelerated TNFα secretion after LPS challenge. Overall, our results indicate that FoxQ1 stimulates monocyte motility, increases pro-inflammatory potential, and directs monocyte migration towards MCP-1 that is crucial for monocyte influx into inflammatory sites. This mechanism could contribute to the pathogenesis of chronic inflammatory disorders such as AD.

The shedded ectodomain of Lyve-1 expressed on M2-like tumor-associated macrophages inhibits melanoma cell proliferation.

Targeting immune cells that support tumor growth is an effective therapeutic strategy in tumor entities such as melanoma. M2-like tumor-associated macrophages (TAM) sustain tumor growth by secreting anti-inflammatory cytokines, proteases and growth factors. In this study, we show that a protein derived from M2-like macrophages namely the shedded ectodomain of Lyve-1 (sLyve-1) decreases human HT144 and murine B16F1 melanoma cell proliferation significantly by acting as a decoy receptor for low-molecular weight hyaluronic acid (LMW-HA) although the LMW-HA/Lyve-1 interaction on lymphatic endothelial cells has been described to induce lymphangiogenesis. This is in line with our finding that the number of LYVE-1+ TAM decreases in higher human melanoma stages and that the early growth of B16 transplant tumors is enhanced in Lyve-1 knockout mice when compared to wild-type mice due to an increased melanoma cell proliferation. LYVE-1 expressing TAM are however true M2 macrophages as they co-express typical M2-markers such as CD163 and CD206. The results of the present study highlight the necessity to carefully determine the net effect particular TAM subpopulations have on tumors before establishing a treatment to target these immune cells.

Fast clonal expansion and limited neural stem cell self-renewal in the adult subependymal zone.

We analyzed the progeny of individual neural stem cells (NSCs) of the mouse adult subependymal zone (SEZ) in vivo and found a markedly fast lineage amplification, as well as limited NSC self-renewal and exhaustion in a few weeks. We further unraveled the mechanisms of neuronal subtype generation, finding that a higher proportion of NSCs were dedicated to generate deep granule cells in the olfactory bulb and that larger clones were produced by these NSCs.

The natural history of incidental meningiomas.

OBJECTIVE: Little information about the natural history of incidental meningiomas exists in the literature. The aim of this study was to determine the natural history of asymptomatic meningiomas by comparing different methods of growth rate calculation to establish a strategy for dealing with these tumors. METHODS: In 47 asymptomatic patients, hospital charts, follow-up records, and imaging studies were reviewed. Of these patients, 6 underwent surgery. Tumor growth rates were determined by calculating the absolute and relative growth rates and the tumor volume doubling times. RESULTS: In 41 patients with conservative management, the average tumor size was 9 cm(3), and the majority (66%) of growth rates were less than 1 cm(3)/yr. The absolute growth rate ranged from 0.03 to 2.62 cm(3)/yr (mean, 0.796 cm(3)/yr). Relative annual growth rates ranged from 0.48 to 72.8% (mean, 14.6%). The tumor doubling time ranged from 1.27 to 143.5 years (mean, 21.6 yr). A moderate correlation between the age and growth rates was found. In young patients, annual growth rates tended to be higher and tumor doubling times shorter. There was no clear correlation between the initial tumor size and tumor doubling time. The mean annual growth rate of meningiomas with calcification was lower than in tumors without calcification. Also, tumors with hypointense or isointense T2 signals on magnetic resonance imaging had a lower growth rate. In the group of six patients with surgical excision, tumor growth rates were higher and tumor doubling times shorter than in the nonsurgical group. CONCLUSION: The majority of incidental meningiomas show minimal growth; thus, they may be observed without surgical intervention unless specific symptoms appear. Tumor growth is associated with patient age. The initial tumor size is not considered a predictive factor for tumor growth. Radiological features, such as calcification or T2 signal intensity, may provide useful information to predict the growth potential of meningiomas.

Expression of stabilin-1 in M2 macrophages in human granulomatous disease and melanocytic lesions.

Stabilin-1 is an endocytotic scavenger receptor, specifically expressed by non-continuous sinusoidal endothelial cells in the liver, spleen and lymph nodes and by M2 or alternatively activated macrophages in human malignancies. We analysed paraffin-embedded tissue of melanocytic lesions and granulomatous diseases for stabilin-1 expression, using the human/murine RS1 antibody. The specificity of the RS1 staining was confirmed in a knockout model, as only M2-like tumor-associated macrophages and vessels of a B16F10 melanoma in wild type mice stained positive; while staining of tumor-associated macrophages and vessels originating from stabilin-1 deficient mice remained negative for stabilin-1 specific antibody RS1. In human specimens, the RS1 antibody stained tumor-associated macrophages in all pathological stages of melanoma. In addition, five cases of juvenile xanthogranulomas and one case of necrobiotic xanthogranuloma were strongly stabilin-1 positive, while Th-1 cytokine dominated granulomatous diseases such as sarcoidosis and granulomatous leprosy were negative. Stabilin-1 positive vessels were found in all analysed non-Langerhans cell histiocytoses and melanocytic lesions. No stabilin-1 positive vessels were present in any other granulomatous diseases.

Prognostic value of immune cell infiltration, tertiary lymphoid structures and PD-L1 expression in Merkel cell carcinomas.

Merkel cell carcinoma (MCC) is an aggressive, virus-associated, neuroendocrine tumor of the skin mainly affecting immunocompromised patients. Higher intratumoral infiltration with CD3 and CD8 positive T-cells is associated with a better prognosis, highlighting the relevance of the immune system for MCC development and progression. In this study 21 primary MCCs were stained with immune cell markers including CD3, CD4, CD8, CD68, CD20, and S100. Furthermore, tumor-infiltrating neutrophils, tertiary lymphoid structures and PD-L1 expression were analyzed and correlated with overall and recurrence free survival. All MCCs were Merkel Cell Polyomavirus positive. Overall and recurrence-free survival did not correlate with intra- and peritumoral CD3 and CD8 T-cell infiltration. In addition, no significant association regarding prognosis was found for tumor-associated neutrophils, tumor-associated macrophages or PD-L1 positivity in MCCs. Interestingly, the presence of tertiary lymphoid structures (TLS) in the tumor microenvironment significantly correlated with recurrence-free survival (P=0.025). In addition, TLS were significantly associated with a higher CD8/CD4 ratio in the tumor periphery (P=0.032), but not in the center of the tumor (P > 0.999). These results demonstrate for the first time that TLS, easily assessed in paraffin-embedded tissue in the tumor periphery of MCCs, may be a valuable prognostic factor indicating prolonged recurrence free survival.