Michael R. Mallmann

Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation

Summary Macrophage activation is associated with profound transcriptional reprogramming. Although much progress has been made in the understanding of macrophage activation, polarization, and function, the transcriptional programs regulating these processes remain poorly characterized. We stimulated human macrophages with diverse activation signals, acquiring a data set of 299 macrophage transcriptomes. Analysis of this data set revealed a spectrum of macrophage activation states extending the current M1 versus M2-polarization model. Network analyses identified central transcriptional regulators associated with all macrophage activation complemented by regulators related to stimulus-specific programs. Applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients. Finally, by integrating murine data from the ImmGen project we propose a refined, activation-independent core signature for human and murine macrophages. This resource serves as a framework for future research into regulation of macrophage activation in health and disease.

High-resolution transcriptome of human macrophages.

Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq) of human macrophages. Using this approach we can now provide a high-resolution transcriptome profile of human macrophages under classical (M1-like) and alternative (M2-like) polarization conditions and demonstrate a dynamic range exceeding observations obtained by previous technologies, resulting in a more comprehensive understanding of the transcriptome of human macrophages. Using this approach, we identify important gene clusters so far not appreciated by standard microarray techniques. In addition, we were able to detect differential promoter usage, alternative transcription start sites, and different coding sequences for 57 gene loci in human macrophages. Moreover, this approach led to the identification of novel M1-associated (CD120b, TLR2, SLAMF7) as well as M2-associated (CD1a, CD1b, CD93, CD226) cell surface markers. Taken together, these data support that high-resolution transcriptome profiling of human macrophages by RNA-seq leads to a better understanding of macrophage function and will form the basis for a better characterization of macrophages in human health and disease.

Unique transcriptome signature of mouse microglia

Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry‐sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types. Microglia and ESdM, both cultured in serum‐free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune‐related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome‐wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

Prognostic significance of tumor-associated macrophages in endometrial adenocarcinoma

OBJECTIVE Endometrial adenocarcinoma is one of the most common gynecologic malignancies worldwide and in stages confined to the uterus considered to have an excellent prognosis. However, in advanced or recurrent cases when surgery fails to achieve disease control other treatment options are less effective. Thus, new therapeutic avenues are needed. METHODS To provide the rationale for the use of novel agents that target immune checkpoints 163 type I endometrial cancer samples were immunohistochemically screened for the presence of CD163(+) tumor-associated macrophages and Foxp3(+) regulatory T cells. Further, a D2-40-based evaluation of lymph vessel density and lymphovascular space invasion was carried out. Correlation analysis with clinicopathological parameters was performed; Kaplan-Meier curves were generated; multivariate analysis was undertaken as appropriate. RESULTS A substantial amount of tumor-associated macrophages and regulatory T cells was detected in all specimens characterizing endometrial cancer as an immunogenic tumor. However, only the increased infiltration of tumor-associated macrophages was proportionally associated with advanced FIGO stages, high tumor grade, increased lymph vessel density, lymphovascular space invasion and lymph node metastasis. Thus, the presence of tumor-associated macrophages indicates aggressive tumor behavior and appeared to be an independent prognostic factor for recurrence-free survival. CONCLUSIONS Our results make future therapeutic approaches that target tumor-associated macrophages reasonable to improve the outcome of women with advanced or recurrent endometrial adenocarcinoma.

Early vs late intervention in twin reversed arterial perfusion sequence

To compare two different management approaches in prenatally diagnosed twin reversed arterial perfusion (TRAP) sequence.

Bronchopulmonary sequestration with massive pleural effusion: pleuroamniotic shunting vs intrafetal vascular laser ablation

To assess the incidence of complications among a relatively large cohort of fetuses with bronchopulmonary sequestration (BPS) and the success of two different intrauterine treatment modalities.

Application of T cell-based transcriptomics to identify three candidate biomarkers for monitoring anti-tgfβr therapy

Objectives The development of targeted drugs would greatly benefit from the simultaneous identification of biomarkers to determine the aspects of bioactivity, drug safety and efficacy, particularly when affecting receptor-signaling pathways. However, the establishment of appropriate systems to monitor drug-induced events requires an accessible surrogate tissue for functional read out. Methods Therefore we present a universal platform based upon T cell-based gene expression profiling for the identification of biomarkers using the antitransforming growth factor β receptor inhibitor LY2109761 as an example. Results Our initial screen revealed 12 candidate genes specifically regulated in T cells by the inhibitor. In subsequent in-vitro and in-vivo analyses, the combined monitoring of independent gene regulation of three genes was established in peripheral blood mononuclear cells as novel pharmacodynamic candidate biomarkers for antitransforming growth factor β receptor based therapies. Conclusion Overall, the proposed concept of biomarker identification can be easily adapted towards other drug candidates for whom gene regulation can be established in cellular components of peripheral blood.

Thoracoamniotic Shunting for Fetal Hydrothorax: Predictors of Intrauterine Course and Postnatal Outcome.

Objective: To assess predictors for survival and complications among a relatively large cohort of fetuses with hydrothorax treated by thoracoamniotic shunting. Methods: All cases with hydrothorax treated by thoracoamniotic shunting in a 10-year period (2002-2011) in two centers were retrospectively reviewed. Results: A total of 78 fetuses with hydrothorax treated with thoracoamniotic shunting were included in the study. Mean gestational age at diagnosis was 25.6 weeks (12-34 weeks). Initial thoracoamniotic shunting was performed at a mean gestational age of 26.5 weeks (16-33 weeks). A mean of 2.53 shunts (1-7) were inserted per fetus. Of the 78 fetuses, 9 (11.5%) died in utero, 69 (88.5%) were born alive and 46 (59%) survived. Prognostic markers significantly associated with nonsurvival were polyhydramnios, hydrops placentae and mediastinal shift at initial scan, onset of hydrops after first shunt placement, rupture of membranes, a shunt-birth interval <4 weeks and low gestational age at birth. In our cohort, fetuses with trisomy 21 had a significantly better survival than euploid fetuses. Conclusions: Although associated with a significant rate of repeated interventions, thoracoamniotic shunting in fetuses with severe hydrothorax results in an overall survival rate of 59%. Fetuses with hydrothorax and trisomy 21 have a better survival when compared to euploid fetuses.

Interval Debulking Surgery in Patients with Federation of Gynecology and Obstetrics (FIGO) Stage IIIC and IV Ovarian Cancer

Background: The feasibility of neoadjuvant chemotherapy (NAC) and the outcome in patients with Federation of Gynecology and Obstetrics (FIGO) IIIC and IV ovarian cancer were assessed. Patients and Methods: 67 patients undergoing interval debulking surgery (IDS) and ≥ 4 courses of platinum-based NAC were analyzed for survival, perioperative morbidity and mortality. Results: The median follow-up was 30 months. The median progression-free survival (PFS) was 17 months, the overall survival (OS) 34 months. The PFS of patients without residual disease (n = 23; 34.3%) was 31 months (p = 0.003), the OS 65 months (p = 0.001). PFS and OS were significantly longer in patients with no residual disease than in patients with 1-10 mm (n = 34; 47.9%) (p = 0.005 and p = 0.0001, respectively) residual disease. No survival benefit was seen for patients with 1-10 mm compared to > 1 cm (n = 12; 16.9%) residual disease (PFS p = 0.518; OS p = 0.077). 1 patient (1.4%) died; 12 patients needed interventional treatment or operation (16.9%) within the first 30 days postoperatively. Out of these, 5 patients (7.0%) had residual or lasting disability. Conclusions: NAC and IDS are safe and feasible in this series of patients with unfavorable prognosis. IDS does not change the goal of complete cytoreduction and therefore does not compensate for a less radical surgical approach.

Omphalocele-Exstrophy-Imperforate Anus-Spinal Defects Complex: Associated Malformations in 12 New Cases

Objective: The omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex is a rare variant of the bladder exstrophy epispadias complex with in most cases unknown etiology. Due to the rarity of the disease, no large series exist that describe the prenatal spectrum of disease or additional malformations. Methods: In this study, we present the prenatal findings in a series of 12 cases. Results: All fetuses showed exstrophy of the bladder, 9/12 omphalocele, 9/12 anal atresia, 10/12 neural tube defects, 4/12 vertebral defects, 5/12 lower extremity defects including clubfeet, and 4/12 a single umbilical artery. Additional malformations included hydrocephalus, hypertelorism, aplasia of the gall bladder, heart defects and kidney malformations. All karyotyped fetuses (11/11) showed a normal karyotype. Conclusions: These findings illustrate the spectrum of disease in prenatal diagnosis.