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Stomach cancer is one of the most common tumors of the gastrointestinal tract. In Russia, stomach cancer is ranked fourth in incidence among malignancies and gives ground for lung, breast and colon cancers. Although the success of PD-1/PD-L1 inhibitors application in clinical practice has been proven in cases of many forms of malignancies, the advantages of application of these types of drugs in cases of stomach cancer has not yet been determined. The purpose of this review is to analyze modern studies concerning the clinical significance of PD-1 and PD-L1 expression in stomach tumors and to find the prospects of immunotherapy these tumors. The large number of studies are currently being described, concerning the analysis of the expression of PD-1, PD-L1 levels in tumor cells and soluble forms of these proteins sPD-1 and sPD-L1 in the serum in patients with stomach cancer. However, their clinical significance is unclear, and in many cases the predictive role of PD-L1 expression for the efficacy of immunotherapy is not yet clearly defined. Further accumulation of knowledge in this area can help to improve the efficacy of existing immunotherapy methods and to develop new predictive criteria.

The data of a comparative enzyme-linked immunosorbent assay of the content of the soluble form of the immunity checkpoint VISTA in the blood serum of 30 healthy donors (control group), 79 patients with primary malignant (osteosarcoma - 30, chondrosarcoma - 31, chordoma - 14) and 14 borderline (giant cell tumor) bone neoplasms are presented. In the general group of patients with malignant neoplasms of bones, the median sVISTA content in blood serum is statistically significant lower than in the control (p = 0.040). In patients with bone tumors and healthy donors over 18 years of age, there was a decrease with age in serum sVISTA levels. There were no significant differences in sVISTA concentration between patients with osteosarcoma, chondrosarcoma and healthy donors. Only in patients with chordoma were sVISTA levels statistically significant lower than in controls (p = 0.013). In the groups of patients with chondrosarcoma and osteosarcoma of the bone, there were no significant associations between the serum sVISTA content and the main clinical and morphological characteristics of the disease. In patients with osteosarcoma, no relationship was found between sVISTA levels and overall survival rates, while in patients with bone chondrosarcoma, there was a tendency towards a favorable prognosis with a high content of the marker in the blood serum.

Esophageal cancer is one of the most aggressive malignant neoplasms, with low survival rates and limited treatment options. In this study we analyzed the microbiome composition and the phenotype of inflammatory tumor infiltrate in squamous cell carcinoma of esophagus (ESCC) and examined possible relationships between them and their prognostic significance. We found that the predominant phyla of microorganisms found in both tumors and adjacent normal tissues were Firmicutes, Proteobacteria, Actinobacteria, Gemmatimonadetes and Bacteroidetes. We established that only bacteria of the genus Staphylococcus differ between tumors and normal tissues. We found a significant correlation between bacterial burden and the phenotype of the tumor stroma. Namely, a group of tumors characterized by a high expression of CD206 (r = −0.3976, p = 0.0056) in the stroma and iNOS (r = −0.2953, p = 0.0439) in tumor cells is characterized by a higher bacterial burden. Further, we established that in the group with a high content of CD206+ macrophages, there is also a predominance of gram-positive bacteria over gram-negative ones. We found that gram-positive bacterial burden is associated with disease prognosis in ESCC showing high content of CD206+ macrophages. In conclusion we established that the tumor microbiome, can be prognostically significant for ESCC when combined with other stromal markers.

In this Special Issue of Biomedicines, we have many insightful reviews and research papers on the subject “Macrophages in Health and Non-infectious Disease”, but first; we should discuss briefly the current situation in the field [...]

CHID1 has been recently described as a predictive marker of different malignant tumors. Thus, monoclonal antibodies (mAbs) for CHID1 detection in different human liquids and in tissues are an important tool for the diagnosis of CHID1-positive cancers. However, only few mAbs have been established to date. In this study we describe the generation of a new hybridoma clone 3D4 producing anti-CHID1 antibodies. 3D4 mAb specifically binds human CHID1 and was successfully used in enzyme-linked immunosorbent assay, immunoblotting, immunofluorescence on paraformaldehyde-fixed cells, and in immunohistochemistry of paraffin-embedded tissue specimens. These results indicate that this new anti-CHID1 mAb 3D4 will be useful in the diagnosis of CHID1-related cancers and is a strong tool for both basic and clinical research on chitinase-like proteins.

This Special Issue (SI) has collected the most recent publications on the mechanisms that macrophages use to regulate homeostasis and their involvement in the pathogenesis of various non-infectious diseases [...]

There is an urgent need for identification of new prognostic markers and therapeutic targets for non-small cell lung cancer (NSCLC). In this study, we evaluated immune cells markers in 100 NSCLC specimens. Immunohistochemical analysis revealed no prognostic value for the markers studied, except CD163 and CD206. At the same time, macrophage markers iNOS and CHID1 were found to be expressed in tumor cells and associated with prognosis. We showed that high iNOS expression is a marker of favorable prognosis for squamous cell lung carcinoma (SCC), and NSCLC in general. Similarly, high CHID1 expression is a marker of good prognosis in adenocarcinoma and in NSCLC in general. Analysis of prognostic significance of a high CHID1/iNOS expression combination showed favorable prognosis with 20 months overall survival of patients from the low CHID1/iNOS expression group. For the first time, we demonstrated that CHID1 can be expressed by NSCLC cells and its high expression is a marker of good prognosis for adenocarcinoma and NSCLC in general. At the same time, high expression of iNOS in tumor cells is a marker of good prognosis in SCC. When used in combination, CHID1 and iNOS show a very good prognostic capacity for NSCLC. We suggest that in the case of lung cancer, tumor-associated macrophages are likely ineffective as a therapeutic target. At the same time, macrophage markers expressed by tumor cells may be considered as targets for anti-tumor therapy or, as in the case of CHID1, as potential anti-tumor agents.

The link between a lung tumor and the lung microbiome is a largely unexplored issue. To investigate the relationship between a lung microbiome and the phenotype of an inflammatory stromal infiltrate, we studied a cohort of 89 patients with non-small cell lung cancer. The microbiome was analyzed in tumor and adjacent normal tissue by 16S rRNA amplicon sequencing. Characterization of the tumor stroma was done using immunohistochemistry. We demonstrated that the bacterial load was higher in adjacent normal tissue than in a tumor (p = 0.0325) with similar patterns of taxonomic structure and alpha diversity. Lung adenocarcinomas did not differ in their alpha diversity from squamous cell carcinomas, although the content of Gram-positive bacteria increased significantly in the adenocarcinoma group (p = 0.0419). An analysis of an inflammatory infiltrate of tumor stroma showed a correlation of CD68, iNOS and FOXP3 with a histological type of tumor. For the first time we showed that high bacterial load in the tumor combined with increased iNOS expression is a favorable prognostic factor (HR = 0.1824, p = 0.0123), while high bacterial load combined with the increased number of FOXP3+ cells is a marker of poor prognosis (HR = 4.651, p = 0.0116). Thus, we established that bacterial load of the tumor has an opposite prognostic value depending on the status of local antitumor immunity.

Background. Tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) contribute significantly to the development of immunosuppressive properties of a tumor. In this study, we performed immunohistochemical analysis of immune cells of esophageal tumors stroma. Methods. Paraffin-embedded tissue specimens from 48 esophageal squamous cell carcinoma (ESCC) patients were retrospectively collected for immunohistochemical analysis of stromal cells. For staining of macrophages, CD68, CD163, CD206, PU.1, and iNOS were used. For T cell detection, CD8, CD3, and FOXP3 were used. Also, we performed staining for PD-L1 that can be expressed on TAMs and tumor cells. Clinicopathological and survival data were collected and analyzed using the χ2 and Fisher exact tests, Kaplan–Meier curves, and the log-rank test. The correlation analysis was performed with Spearman’s rank correlation coefficient. Results. We found that FOXP3 expression was associated with age (p=0.042) and iNOS expression was associated with the disease stage (p=0.044). In addition, FOXP3 and CD163 appeared to be markers of good prognosis (HR=0.4420, p=0.0325, and HR=0.4447, p=0.0456, respectively). Significant association between PU.1+ and CD68+ macrophages (r=0.833; p≤0.001) and between PU.1+ and CD163+ macrophages (r=0.500; p≤0.001) was established; positive association between PU.1 and CD206 expression was also observed (r=0.250; p=0.043). Conclusions. Large amounts of CD163+ macrophages and FOXP3+ Т cells appear to be markers of good prognosis of ESCC. The number of PU.1+ macrophages strongly correlates with the number of CD68+ macrophages; therefore, usage of PU.1 as a potential macrophage marker can be recommended for esophageal tumors.

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.

Recent research on cancer-associated microbial communities led to the accumulation of data on the interplay between bacteria, immune and tumor cells, the pathways of bacterial induction of carcinogenesis, and its meaningfulness for medicine. Microbial communities that have any kind of impact on tumor progression and microorganisms associated with tumors have been defined as oncobiome. Over the last decades, a number of studies were dedicated toHelicobacter pyloriand its role in the progression of stomach tumors, so this correlation can be regarded as proven. Involvement of bacteria in the induction of lung cancer has been largely ignored for a long time, though some correlations between this type of cancer and lung microbiome were established. Despite the fact that in the present the microbial impact on lung cancer progression has many confirmations, the underlying mechanisms are poorly understood. Microorganisms can contribute to tumor initiation and progression through production of bacteriotoxins and other proinflammatory factors. The purpose of this review is to organize the available data on lung cancer microbiome and its role in malignant tumor progression.

BACKGROUND The nucleotide excision repair (NER) pathway, defective in xeroderma pigmentosum (XP) patients, removes DNA photolesions in order to prevent carcinogenesis. Complementation group C (XP-C) is the most frequent group of XP patients worldwide. METHODS We analyzed seven XP-C patients clinically and molecular-genetically applying: post-UV cell survival (MTT-assay), quantitative Real-time PCR, sequencing on chromosomal as well as cDNA level, and in silico interpretation of sequencing data. RESULTS All cases displayed diminished post-UV cell survival as well as reduced XPC mRNA levels. Five homozygous and two heterozygous disease causing mutations were identified. A large chromosomal deletion of ~5.8 kb identified in XP174MA leads to an unique in frame deletion of XPC exon 2 and exon 3. In silico analysis revealed the deletion of 102 amino acids in the N-terminal part of XPC while leaving the C-terminal domain intact. The novel c.361delA mutation in XP168MA leads to a frameshift in exon 3 resulting in a premature stop codon 27 codons downstream of the deleted adenine. CONCLUSION Our analysis confirms that XP-C patients without increased sun sensitivity develop non-melanoma skin cancers earlier than sun-sensitive XP-C patients. Reduced cellular mRNA levels are characteristic for XP complementation group C and qRT-PCR represents a rapid diagnostic tool.

Stabilin-1 is a multifunctional scavenger receptor expressed on alternatively-activated macrophages. Stabilin-1 mediates phagocytosis of "unwanted-self" components, intracellular sorting, and endocytic clearance of extracellular ligands including SPARC that modulates breast cancer growth. The expression of stabilin-1 was found on tumor-associated macrophages (TAM) in mouse and human cancers including melanoma, lymphoma, glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphoma the expression and functional role of stabilin-1 in breast cancer was unknown. Here, we demonstrate that stabilin-1 is expressed on TAM in human breast cancer, and its expression is most pronounced on stage I disease. Using stabilin-1 knockout (ko) mice we show that stabilin-1 facilitates growth of mouse TS/A mammary adenocarcinoma. Endocytosis assay on stabilin-1 ko TAM demonstrated impaired clearance of stabilin-1 ligands including SPARC that was capable of inducing cell death in TS/A cells. Affymetrix microarray analysis on purified TAM and reporter assays in stabilin-1 expressing cell lines demonstrated no influence of stabilin-1 expression on intracellular signalling. Our results suggest stabilin-1 mediated silent clearance of extracellular tumor growth-inhibiting factors (e.g. SPARC) as a mechanism of stabilin-1 induced tumor growth. Silent clearance function of stabilin-1 makes it an attractive candidate for delivery of immunomodulatory anti-cancer therapeutic drugs to TAM.

Low-grade chronic inflammation underlies the development of the most dangerous cardiometabolic disorders including type 2 diabetes and its vascular complications. In contrast to acute inflammation induced by bacteria and viruses, chronic inflammation can be driven by abnormal reaction to endogenous factors, including Th2 cytokines, metabolic factors like advanced glycation end products (AGEs), modified lipoproteins, or hyperglycemia. The key innate immune cells that recognize these factors in blood circulation are monocytes. Inflammatory programming of monocytes which migrate into tissues can, in turn, result into generation of tissue macrophages with pathological functions. Therefore, determination of the molecular and functional phenotype of circulating monocytes is a very promising diagnostic tool for the identification of hidden inflammation, which can precede the development of the pathology. Here we propose a new test system for the identification of inflammatory programming of monocytes: surface biomarkers and ex vivo functional system. We summarize the current knowledge about surface biomarkers for monocyte subsets, including CD16, CCR2, CX3CR1, CD64, stabilin-1 and CD36, and their association with inflammatory human disorders. Furthermore, we present the design of an ex vivo monocyte-based test system with minimal set of parameters as a potential diagnostic tool for the identification of personalized inflammatory responses.

Major problems with biomedical devices in particular implants located in nonsterile environments concern: (i) excessive immune response to the implant, (ii) development of bacterial biofilms, and (iii) yeast and fungi infections. An original multifunctional coating that addresses all these issues concomitantly is developed. A new exponentially growing polyelectrolyte multilayer film based on polyarginine (PAR) and hyaluronic acid (HA) is designed. The films have a strong inhibitory effect on the production of inflammatory cytokines released by human primary macrophage subpopulations. This could reduce potential chronic inflammatory reaction following implantation. Next, it is shown that PAR, due to its positive charges, has an antimicrobial activity in film format against Staphylococcus aureus for 24 h. In order to have a long-term antimicrobial activity, a precursor nanoscale silver coating is deposited on the surface before adding the PAR/HA films. Moreover, the PAR/HA films can be easily further functionalized by embedding antimicrobial peptides, like catestatin (CAT), a natural host defense peptide. This PAR/HA+CAT film proves to be effective as an antimicrobial coating against yeast and fungi and its cytocompatibility is also assessed. Finally, this all-in-one system constitutes an original strategy to limit inflammation and prevents bacteria, yeast, and fungi infections.

In recent years, the new direction such as identification of informative circulating markers reflecting molecular genetic changes in the DNA of tumor cells was actively developed. Smoking-related DNA adducts are very promising research area, since they indicate high pathogenetic importance in the lung carcinogenesis and can be identified in biological samples with high accuracy and reliability using highly sensitive mass spectrometry methods (TOF/TOF, TOF/MS, MS/MS). The appearance of DNA adducts in blood or tissues is the result of the interaction of carcinogenic factors, such as tobacco constituents, and the body reaction which is determined by individual characteristics of metabolic and repair systems. So, DNA adducts may be considered as a cumulative mirror of heterogeneous response of different individuals to smoking carcinogens, which finally could determine the risk for lung cancer. This review is devoted to analysis of the role of DNA adducts in lung carcinogenesis in order to demonstrate their usefulness as cancer associated markers. Currently, there are some serious limitations impeding the widespread use of DNA adducts as cancer biomarkers, due to failure of standardization of mass spectrometry analysis in order to correctly measure the adduct level in each individual. However, it is known that all DNA adducts are immunogenic, their accumulation over some threshold concentration leads to the appearance of long-living autoantibodies. Thus, detection of an informative pattern of autoantibodies against DNA adducts using innovative multiplex ELISA immunoassay may be a promising approach to find lung cancer at an early stage in high-risk groups (smokers, manufacturing workers, urban dwellers).

AMACR (alpha-methylacyl-CoA racemase) has been recently described as a prostate cancer-specific gene that encodes a protein involved in the beta-oxidation of branched chain fatty acids. Expression of AMACR protein is found in prostatic adenocarcinoma, but not in benign prostatic tissue. Thus, monoclonal antibodies (mAbs) for AMACR detection are an important tool for the diagnosis of AMACR-positive cancers. However, only a few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we describe the generation of a new hybridoma clone G8 producing anti-AMACR antibodies. G8 mAb specifically binds human AMACR and was successfully used in immunoblotting and immunofluorescence on paraformaldehyde-fixed cells and in IHC of paraffin-embedded tumor specimens. These results indicate that this new anti-AMACR mAb G8 would be useful in the diagnosis of AMACR-related cancers and would be a strong tool in both basic and clinical research on AMACR.

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.

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytoldne profile that can support of diabetes-associated inflammation and development of vascular complications. (C) 2016 Elsevier GmbH. All rights reserved.

Only 16 XPG-defective patients with 20 different mutations have been described. The current hypothesis is that missense mutations impair repair (xeroderma pigmentosum (XP) symptoms), whereas truncating mutations impair both repair and transcription (XP and Cockayne syndrome (CS) symptoms). We identified three cell lines of XPG-defective patients (XP40GO, XP72MA, and XP165MA). Patients' fibroblasts showed a reduced post-UVC cell survival. The reduced repair capability, assessed by host cell reactivation, could be complemented by XPG cDNA. XPG mRNA expression of XP165MA, XP72MA, and XP40GO was 83%, 97%, and 82.5%, respectively, compared with normal fibroblasts. XP165MA was homozygous for a p.G805R mutation; XP72MA and XP40GO were both compound heterozygous (p.W814S and p.E727X, and p.L778P and p.Q150X, respectively). Allele-specific complementation analysis of these five mutations revealed that p.L778P and p.W814S retained considerable residual repair activity. In line with the severe XP/CS phenotypes of XP72MA and XP165MA, even the missense mutations failed to interact with the transcription factor IIH subunits XPD and to some extent cdk7 in coimmunoprecipitation assays. Immunofluorescence techniques revealed that the mutations destabilized early recruitment of XP proteins to localized photodamage and delayed their redistribution in vivo. Thus, we identified three XPG missense mutations in the I-region of XPG that impaired repair and transcription and resulted in severe XP/CS.

Patients belonging to xeroderma pigmentosum (XP) complementation group C comprise one-third of all XP patients. Only four major reports compiled larger groups of XP-C patients from southern Europe (12 pts), North America (16 pts) and Africa (14 and 56 pts) as well as their genetic background (46 XPC mutations). We identified 16 XP-C patients from Germany. Interestingly, only five patients exhibited severe sun sensitivity. The mean age of XP diagnosis was 9.4 years, and the median age of the first skin cancer was 7 years. Neurological symptoms were absent in all but two patients. Primary fibroblasts from all 16 patients showed reduced post-UV cell survival (mean: 50% vs 93% in normal cells) and reduced reactivation of an UV-treated luciferase reporter gene (mean: 6.4% vs 30.7% in normal cells). XPC mRNA expression was also greatly reduced compared with normal cells (mean: 14.3%; range 8.325.7%) except in XP47MA (274.1%). All patients carried homozygous XPC mutations. Four mutations have been described previously: c.1747_1748delTG (found in 4/16), c.567 C>T (4/16), c.1839 C>T (1/16) and a complex insertion/deletion mutation in exon 9 (1/16). The novel frameshift mutations c.446_447delAG (2/16), c.1525insA (1/16) and c.2271delC (1/16) lead to truncated XPC proteins as does the novel nonsense mutation c.843C>T (1/16). XP47MA carries an interesting mutation (c.2538_2540delATC; p.Ile812del) resulting in an in-frame single amino acid deletion. This mutation results in a classical XP phenotype, a non-functional XPC protein, but elevated XPC mRNA expression. Our study indicates that extrinsic factors may contribute to XP-C symptom severity due to nonsense-mediated message decay.

Tumor associated macrophages (TAMs) are an important element of tumor stroma. They originate from blood monocytes attracted by chemokines and cytokines produced by tumor cells and, being instructed by tumor microenvironment, develop into potent tumor-supporting cell population. TAMs were demonstrated to directly stimulate tumor cell proliferation and to promote angiogenesis. Further TAMs provide for efficient immune escape by producing immunosuppressive cytokines and facilitate tumor dissemination by producing extracellular matrix remodeling enzymes. In renal cell carcinoma (RCC), numerous studies were performed for elucidation of the role of TAM in tumor progression. Using pan-macrophages marker CD68 and type 2 macrophage (M2) markers CD163 and CD206, it was demonstrated that increased density of TAMs is associated with poor survival of patients. Although most of the studies are focused on M2 population in RCC, several markers rather typical for type 1 macrophages (M1) were also characterized. Macrophages isolated from RCC tumors were shown to produce proinflammatory cytokines TNFα, IL-1β, IL-6, and CCL2. It can be concluded that RCC is an excellent example of a tumor with hybrid phenotype of TAMs that share both M1 and M2 properties. Moreover, TAMs seem to be an attractive therapeutic target as well. Further investigations are needed for identification of RCC-specific TAM markers with high predictive capacity and/or suitable for therapeutic targeting.

IL17RB is the receptor for IL17E, the only member of IL17 family promoting Th2 reactions. The mechanism of IL17BR regulation is poorly understood. We previously demonstrated that expression of IL17RB is induced on human macrophages by IL4 and enhanced by TGFβ. In the present study we investigated the immediate signaling targets of IL17RB. Using Yeast Two Hybrid screening we identified DAZAP2 as a binding partner of IL17RB. We established that 2 SH2-binding domains of DAZAP2 are essential for its binding to IL17RB. Deletion of these domains or substitution of tyrosines to alanines abrogates the binding. In IL17RB DAZAP2-binding domain was mapped to the region between aa 329 and 347 within its cytoplasmic part. Confocal microscopy revealed that in primary human macrophages that do not express IL17RB DAZAP2 is predominantly localized in the nucleus, while in IL17RB positive macrophages a portion of DAZAP2 is visualized in the cytoplasm. Stimulation of IL17RB with its ligand IL17E induces accumulation of DAZAP2 in the cytoplasm. Further we established that DAZAP2 interacts with Smurf2 an E3 ubiquitin ligase which induces proteasome-dependent degradation of the protein. In summary we established a new mechanism of IL17RB regulation-Smurf2 dependent degradation of its adaptor protein DAZAP2.

Objectives We characterized the transcriptional profiles of GM-CSF- (GM-MO) and M-CSF-induced macrophages (M-MO) and investigated in situ a subset of differentially expressed genes in human and mouse atherosclerotic lesions. Methods and results Using microarrays we identified a number of genes and biological processes differentially regulated in M-MO vs GM-MO. By varying in culture the M-CSF/GM-CSF ratio (0–10), a spectrum of macrophage phenotypes was explored by RT-QPCR. M-CSF (10ng/ml) stimulated expression of several genes, including selenoprotein-1 ( SEPP1 ), stabilin-1 ( STAB1 ) and CD163 molecule-like-1 ( CD163L1 ) which was inhibited by a low dose of GM-CSF (1ng/ml); M-CSF inhibited the expression of pro-platelet basic protein ( PPBP ) induced by GM-CSF. Combining Tissue Microarrays/quantitative immunohistochemistry of human aortic lesions with RT-QPCR expression data either from human carotids vs mammary non-atherosclerotic arteries or from the apoE null mice normal and atherosclerotic aortas showed that, STAB1 , SEPP1 and CD163L1 (M-CSF-sensitive genes) and PPBP (GM-CSF-sensitive gene) were expressed in both human arterial and apoE null mice atherosclerotic tissues. Conclusion A balance between M-CSF vs GM-CSF defines macrophage functional polarisation and may contribute to the progression of atherosclerosis.

Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.

Extraneuronal acetylcholine (ACh) has been demonstrated to influence a plethora of cutaneous cell functions in an autocrine, paracrine and endocrine fashion. Previously, we could demonstrate a differentiation-specific expression of its nicotinic (nAChR) and muscarinic receptors (mAChR) in human epidermis and its adnexal structures including sebaceous glands. Using an immortalized human sebaceous gland cell line (SZ95 sebocytes), we examined the AChR expression pattern in vitro. In proliferating and confluent SZ95 sebocytes, a wide range of AChR subunits could be detected using RT-PCR. In particular, we detected mRNA coding for the α3, α5, α6, α7, α10, β1, β2 and β4 nAChR as well as the M1, and M3−5. The α1 and M2 and M5 subunits could only be detected in confluent SZ95 sebocytes, while in proliferating ones these subunits remained negative. The α2, α4, α9 and β3 nAChR could not be detected. Using functional assays, we assessed the impact of cholinergic agonists and antagonists on sebocyte proliferation and differentiation as evidenced by lipid production. Atropine (inhibition of all mAChR) and himbacine (inhibition of M2 and M4) potently inhibited SZ95 sebocyte proliferation in a dose-dependent manner with a maximum effect at milimolar concentrations, while glycopyrrolate (inhibition of M1 and M3) inhibited proliferation only at high concentrations (100 µM). Interestingly also muscarine inhibited SZ95 sebocyte proliferation, however, with a maximum effect (50%) at nanomolar concentrations. The inhibitory effect of mecamylamine was less pronounced (20%). Nicotine strongly induced SZ95 sebocyte proliferation to more than 200% in a dose-dependent manner. Lipid production was increased by nicotine and muscarine in the micromolar range. Inhibition of nAChR by mecamylamine did not significantly influence lipid production, while even nanomolar concentrations of atropine significantly increased lipid production. In conclusion, we could demonstrate highly potent effects of the cholinergic system on sebocyte proliferation and lipid production in vitro, mediated by wealth of different AChR subunits present at least at the mRNA level. In particular, promotion of sebocyte proliferation seems to be an attractive explanation for the exacerbation of sebaceous gland–related disorders like acne under the influence of chronic nicotine ingestion. In addition, seborrhea observed after treatment with anticholinergic drugs is well in line with an increase in lipid production that we found after treatment of sebocytes with atropine.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 μg/ml in maternal circulation and stays below 0.5 μg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.

Tumour associated macrophages (TAM) represent an important component of tumour stroma. They develop under the influence of tumour microenvironment where transforming growth factor (TGF)β is frequently present. Activities of TAM regulated by TGFβ stimulate proliferation of tumour cells and lead to tumour immune escape. Despite high importance of TGFβ-induction of TAM activities till now our understanding of the mechanism of this induction is limited. We have previously developed a model of type 2 macrophages (M2) resembling certain properties of TAM. We established that in M2 TGFβRII is regulated on the level of subcellular sorting by glucocorticoids. Further studies revealed that in M2 with high levels of TGFβRII on the surface TGFβ activates not only its canonical Smad2/3-mediated signaling, but also Smad1/5-mediated signaling, what is rather typical for bone morphogenetic protein (BMP) stimulation. Complexity of macrophage populations, however, allows assumption that TGFβ signalling may function in different ways depending on the functional state of the cell. To understand the peculiarities of TGFβ signalling in human TAMs experimental systems using primary cells have to be developed and used together with the modern mathematical modelling approaches.

The aim of this study was to analyze the influence of cholinergic and anticholinergic drugs on epidermal physiology using organotypic cocultures (OTCs). Blocking of all acetylcholine receptors (AChRs) by combined treatment with mecamylamine and atropine or treatment with strychnine (blocking alpha9nAChR) for 7-14 days resulted in a complete inhibition of epidermal differentiation and proliferation. Blockage of nicotinic (n)AChR with mecamylamine led to a less pronounced delay in epidermal differentiation and proliferation than blockage of muscarinic (m)AChR with atropine, evidenced by reduced epithelial thickness and expression of terminal differentiation markers like cytokeratin 2e or filaggrin. In OTCs treated with atropine, mecamylamine, or strychnine, we could demonstrate intracellular lipid accumulation in the lower epidermal layers, indicating a severely disturbed epidermal barrier. In addition, we observed prominent acantholysis in the basal and lower suprabasal layers in mecamylamine-, atropine-, and strychnine-treated cultures, accompanied by a decreased expression of cell adhesion proteins. This globally reduced cell adhesion led to cell death via intrinsic activation of apoptosis. In contrast, stimulation of nAChR and mAChR with cholinergic drugs resulted in a significantly thickened epithelium, accompanied by an improved epithelial maturation. In summary, we show that epidermal AChR are crucially involved in the regulation of epidermal homeostasis.

Macrophages (Mphi) represent a dynamic cell population that develops and operates within a changing microenvironment. In parallel to Th1/Th2 cells, primary Mphi may undergo classical (Mphi1) or alternative (Mphi2) activation. Here, we investigated whether Mphi1/Mphi2 may be re-polarized by a secondary stimulation by Th1 or Th2 cytokines or by exogenous danger signals. We established that Mphi1IFNgamma respond to alternative activation by IL-4 and IL-10 by de novo secretion of Th2 cytokines AMAC-1 and IL-1ra, and by an increase in phagocytic capacity and a decrease in bactericidal activity. Vice versa, Mphi2 responded to classical activation by IFNgamma exhibiting reduced phagocytosis and significantly increased bacterial killing while being refractory regarding secretion of TNFalpha, IL-1beta and IL-12. In response to the bacterial danger signals LPS and MDP, both Mphi1 and Mphi2 produced IL-1beta and TNFalpha; in addition Mphi2 expressed the Th1-inducing cytokine IL-12. The ability of Mphi to be re-polarized and to react to exogenous danger signals is a precondition to down-regulate an outdated immune reaction and to retain the capacity to mount an adequate anti-bacterial response. Selective refractoriness of Mphi1 and Mphi2 to IFNgamma- and LPS-induced cytokine secretion may contribute to prevent autoimmunity.