Expression of free fatty acid receptor 2 in normal and neoplastic tissues.

OBJECTIVE: Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study was to comprehensively characterise the expression profile of FFAR2 in a large series of human normal and neoplastic tissues using immunohistochemistry thus providing a basis for further in-depth investigations into its potential diagnostic or therapeutic importance. METHODS: We developed a novel rabbit polyclonal anti-FFAR2 antibody, 0524, directed against the C-terminal region of human FFAR2. Antibody specificity was confirmed via Western blot analyses and immunocytochemistry using the FFAR2-expressing cell line BON-1 and FFAR2-specific small interfering RNA as well as native and FFAR2-transfected HEK-293 cells. The antibody was then used for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic human tissues. RESULTS: In normal tissues, FFAR2 was mainly present in distinct cell populations of the cerebral cortex, follicular cells and C cells of the thyroid, cardiomyocytes of the heart, bronchial epithelia and glands, hepatocytes and bile duct epithelia of the liver, gall bladder epithelium, exocrine and ß-cells of the endocrine pancreas, glomerular mesangial cells and podocytes as well as collecting ducts of the kidney, intestinal mucosa (particularly enteroendocrine cells), prostate epithelium, seminiferous tubules of the testicles, and placental syncytiotrophoblasts. In neoplastic tissues, FFAR2 was particularly prevalent in papillary thyroid carcinomas, parathyroid adenomas, and gastric, colon, pancreatic, hepatocellular, cholangiocellular, urinary bladder, breast, cervical, and ovarian carcinomas. CONCLUSIONS: We generated and characterised a novel rabbit polyclonal anti-human FFAR2 antibody that is well-suited for visualising FFAR2 expression in human routine pathology tissues. This antibody is also suitable for Western blot and immunocytochemistry experiments. To our knowledge, this antibody enabled the first broad FFAR2 protein expression profile in various normal and neoplastic human tissues.

Expression prevalence and dynamics of GPCR somatostatin receptors 2 and 3 as cancer biomarkers beyond NET: a paired immunohistochemistry approach.

Somatostatin receptors are clinically validated GPCR biomarkers for diagnosis and treatment of various neuroendocrine tumors (NET). Among the five somatostatin receptors, SST2 and SST3 are associated with apoptosis and cell cycle arrest, making these receptor subtypes better differentiated targets in precision oncology. In this study we performed immunohistochemistry of paired tissue microarrays containing 1125 cores, representing 43 tumor types, each stained for SST2 and SST3. A 12-point immunoreactive scoring (IRS) range was used for interpretation of the staining results. We analyzed the results twice, using the conventional positivity IRS cutoffs ≥ 3 and more stringent ≥ 6. Evaluation of receptors expression dynamics was performed for tumor-nodes-metastases (TNM) defined subgroups (ovarian and hepatocellular adenocarcinomas) as a function of their tumor stage. Our results indicate that two-thirds of tested cores exhibit clinically significant expression of at least SST2 or SST3 (IRS ≥ 6). The expression prevalence of both receptors tends to decline with tumor progression. However, an unexpected upregulation of both SST2 and SST3 reemerged in metastases suggesting conserved receptors genetic potential during tumor life cycle. We suggest that SST2 and SST3 should be further explored as potential biomarkers and therapeutic tools for maximizing the efficiency of somatostatin-based precision oncology of solid tumors beyond NET.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues.

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Sodium thiosulfate refuels the hepatic antioxidant pool reducing ischemia-reperfusion-induced liver injury.

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na2S, fast releasing) and sodium thiosulfate (STS, Na2S2O3, slow releasing). The mitoprotection and toxicity of STS and Na2S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1 h followed by reperfusion of 1 or 24 h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24 h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity.

Expression of the Calcitonin Receptor-like Receptor (CALCRL) in Normal and Neoplastic Tissues.

Little information is available concerning protein expression of the calcitonin receptor-like receptor (CALCRL) at the protein level. Here, we developed a rabbit monoclonal antibody, 8H9L8, which is directed against human CALCRL but cross-reacts with the rat and mouse forms of the receptor. We confirmed antibody specificity via Western blot analyses and immunocytochemistry using the CALCRL-expressing neuroendocrine tumour cell line BON-1 and a CALCRL-specific small interfering RNA (siRNA). We then used the antibody for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic tissues. In nearly all tissue specimens examined, CALCRL expression was detected in the capillary endothelium, smooth muscles of the arterioles and arteries, and immune cells. Analyses of normal human, rat, and mouse tissues revealed that CALCRL was primarily present in distinct cell populations in the cerebral cortex; pituitary; dorsal root ganglia; epithelia, muscles, and glands of the larger bronchi; intestinal mucosa (particularly in enteroendocrine cells); intestinal ganglia; exocrine and endocrine pancreas; arteries, capillaries, and glomerular capillary loops in the kidneys; the adrenals; Leydig cells in the testicles; and syncytiotrophoblasts in the placenta. In the neoplastic tissues, CALCRL was predominantly expressed in thyroid carcinomas, parathyroid adenomas, small-cell lung cancers, large-cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear-cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours with strong expression of CALCRL, the receptor may represent a useful target structure for future therapies.

Periportal steatosis in mice affects distinct parameters of pericentral drug metabolism.

Little is known about the impact of morphological disorders in distinct zones on metabolic zonation. It was described recently that periportal fibrosis did affect the expression of CYP proteins, a set of pericentrally located drug-metabolizing enzymes. Here, we investigated whether periportal steatosis might have a similar effect. Periportal steatosis was induced in C57BL6/J mice by feeding a high-fat diet with low methionine/choline content for either two or four weeks. Steatosis severity was quantified using image analysis. Triglycerides and CYP activity were quantified in photometric or fluorometric assay. The distribution of CYP3A4, CYP1A2, CYP2D6, and CYP2E1 was visualized by immunohistochemistry. Pharmacokinetic parameters of test drugs were determined after injecting a drug cocktail (caffeine, codeine, and midazolam). The dietary model resulted in moderate to severe mixed steatosis confined to periportal and midzonal areas. Periportal steatosis did not affect the zonal distribution of CYP expression but the activity of selected CYPs was associated with steatosis severity. Caffeine elimination was accelerated by microvesicular steatosis, whereas midazolam elimination was delayed in macrovesicular steatosis. In summary, periportal steatosis affected parameters of pericentrally located drug metabolism. This observation calls for further investigations of the highly complex interrelationship between steatosis and drug metabolism and underlying signaling mechanisms.

Co-Expression of Adaptor Protein FAM159B with Different Markers for Neuroendocrine Cells: An Immunocytochemical and Immunohistochemical Study.

Little is known about the adaptor protein FAM159B. Recently, FAM159B was shown to be particularly expressed in neuroendocrine cells and tissues, such as pancreatic islets and neuroendocrine cells of the bronchopulmonary and gastrointestinal tracts, as well as in different types of neuroendocrine tumours. To gain insights into possible interactions of FAM159B with other proteins and/or receptors, we analysed the co-expression of FAM159B and various neuroendocrine-specific markers in the cancer cell lines BON-1, PC-3, NCI-h82, OH-1, and A431 and also in human pancreatic tissues and pancreatic neuroendocrine tumours. The markers included prominent markers of neuroendocrine differentiation, such as chromogranin A (CgA), neuron-specific enolase (NSE), synaptophysin (SYP), insulinoma-associated protein 1 (INSM1), neural cell adhesion molecule 1 (NCAM1), serotonin (5-HT), somatostatin-14/28 (SST), and several receptors that are typically expressed by neuroendocrine cells, such as dopamine receptor 2 (D2R), somatostatin receptor (SSTR) 1, 2, 3, 4 and 5, and regulator of G-protein signalling 9 (RGS9). FAM159B was expressed evenly throughout the cytosol in all five cancer cell lines. Immunocytochemical and immunohistochemical analyses revealed co-expression of FAM159B with SYP, INSM1, RGS9, D2R, SSTR2, SSTR3, SSTR4, and SSTR5 and strong overlapping co-localisation with NSE. Double-labelling and co-immunoprecipitation Western blot analyses confirmed a direct association between FAM159B and NSE. These results suggest the involvement of FAM159B in several intracellular signalling pathways and a direct or indirect influence on diverse membrane proteins and receptors.

Evaluation of PD-L1 expression in a large set of gastroenteropancreatic neuroendocrine tumours and correlation with clinicopathological data.

BACKGROUND: Targeting programmed death protein 1 (PD-1) or its ligand PD-L1 is a promising therapeutic approach for many types of cancer in which PD-L1 is overexpressed. However, data on PD-L1 expression levels in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are limited and contradictory. METHODS: We evaluated PD-L1 expression in 457 archived, formalin-fixed, paraffin-embedded GEP-NEN samples from 175 patients by immunohistochemistry using the highly sensitive monoclonal anti-PD-L1 antibody 73-10. The immunostaining was semiquantitatively evaluated using a 12-point immunoreactivity score (IRS) taking both PD-L1-positive tumour cells and immune cells into account. Tumour samples with an IRS ≥ 3 were considered PD-L1-positive. Results were correlated with clinicopathological data and with the expression of several typical markers and receptors for neuroendocrine tumours. RESULTS: Of the GEP-NEN samples, 73% were PD-L1-positive. The median IRS value across all samples was 4.0, corresponding to low expression. PD-L1 immunostaining was predominantly localised at the plasma membrane of the tumour cells. Positive correlations were observed between PD-L1 expression and tumour grading or Ki-67 index, between PD-L1 expression and the expression of chromogranin A, and between PD-L1 expression and the expression of each of the five somatostatin receptors. PD-L1 expression was lower in tumours with lymph node metastases at diagnosis than in those without regional metastasis and lower in high-stage than in earlier-stage tumours. No association was noted between PD-L1 expression and patient survival. CONCLUSIONS: PD-L1 expression is common in GEP-NENs and increases with malignancy. Therefore, especially in high-grade GEP-NENs, targeting the PD-1/PD-L1 axis could be a promising additional therapeutic strategy.

Reassessment of somatostatin receptor SST4 expression in bronchopulmonary and gastroenteropancreatic neuroendocrine neoplasms using the novel rabbit monoclonal anti-human SST4 antibody 7H49L61.

Somatostatin receptors SST1, SST2, and SST5 are overexpressed in neuroendocrine neoplasms (NENs), but little is known about SST4 expression in NENs because of a lack of specific monoclonal antibodies. We recently developed and thoroughly characterised a rabbit monoclonal anti-human SST4 antibody, 7H49L61, and showed that it is well suited for identifying SST4 expression in routine pathology samples. The present study aimed to re-evaluate SST4 expression in a large set of NEN samples using this antibody. For this purpose, we assessed SST4 expression in 722 formalin-fixed, paraffin-embedded NEN samples from 274 patients by immunohistochemistry using the novel antibody 7H49L61. The immunostaining was semiquantitatively evaluated using the 12-point immunoreactivity score (IRS), and the results were correlated with clinicopathological data. SST4 was detected in 39.3% of all NENs, but with a median IRS of 2.0, its expression intensity was negligible overall. In all cases, both cytoplasmic and membraneous staining was observed. SST4 expression was somewhat higher in bronchopulmonary NEN (BP-NEN) than in gastroenteropancreatic NEN (GEP-NEN) but still very low. SST4 expression positively correlated with favourable patient outcomes in BP-NEN but had a positive association with Ki-67 index or tumour grading and a negative interrelationship with overall survival in GEP-NEN. In conclusion, unlike that of other SST subtypes, SST4 expression in both BP-NEN and GEP-NEN is negligible and of no diagnostic or therapeutic relevance.

Comparative evaluation of somatostatin and CXCR4 receptor expression in different types of thyroid carcinoma using well-characterised monoclonal antibodies.

BACKGROUND: Papillary and follicular thyroid carcinomas can be treated surgically and with radioiodine therapy, whereas therapeutic options for advanced stage IV medullary and for anaplastic tumours are limited. Recently, somatostatin receptors (SSTs) and the chemokine receptor CXCR4 have been evaluated for the treatment of thyroid carcinomas, however, with contradictory results. METHODS: The expression of the five SSTs and of CXCR4 was assessed in 90 samples from 56 patients with follicular, papillary, medullary, or anaplastic thyroid carcinoma by means of immunohistochemistry using well-characterised monoclonal antibodies. The stainings were evaluated using the Immunoreactivity Score (IRS) and correlated to clinical data. In order to further substantiate the immunohistochemistry results, in serial sections of a subset of the samples receptor expression was additionally examined at the mRNA level using qRT-PCR. RESULTS: Overall, SST and CXCR4 protein expression was low in all four entities. In single cases, however, very high IRS values for SST2 and CXCR4 were observed. SST2 was the most frequently expressed receptor, found in 38% of cases, followed by SST5 and SST4, found in 14 and 9% of tumours, respectively. SST1 and SST3 could not be detected to any significant extent. CXCR4 was present in 12.5% of medullary and 25% of anaplastic carcinomas. Expression SST3, SST4, SST5 and CXCR4 was positively correlated with expression of the proliferation marker Ki-67. Additionally, a negative interrelationship between SST4 or SST5 expression and patient survival and a positive association between SST3 expression and tumour diameter were observed. qRT-PCR revealed a similar receptor expression pattern to that seen at the protein level. However, probably due to the low overall expression, no correlation was found for the SSTs or the CXCR4 between the IRS and the mRNA values. CONCLUSIONS: SST- or CXCR4-based diagnostics or therapy in thyroid carcinomas should not be considered in general but may be feasible in single cases with high levels of expression of these receptors.

Assessment of G Protein-Coupled Oestrogen Receptor Expression in Normal and Neoplastic Human Tissues Using a Novel Rabbit Monoclonal Antibody.

In addition to the classical oestrogen receptors, ERα and ERß, a G protein-coupled oestrogen receptor (GPER) has been identified that primarily mediates the rapid, non-genomic signalling of oestrogens. Data on GPER expression at the protein level are contradictory; therefore, the present study was conducted to re-evaluate GPER expression by immunohistochemistry to obtain broad GPER expression profiles in human non-neoplastic and neoplastic tissues, especially those not investigated in this respect so far. We developed and thoroughly characterised a novel rabbit monoclonal anti-human GPER antibody, 20H15L21, using Western blot analyses and immunocytochemistry. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded human tissue samples. In normal tissue, GPER was identified in distinct cell populations of the cortex and the anterior pituitary; islets and pancreatic ducts; fundic glands of the stomach; the epithelium of the duodenum and gallbladder; hepatocytes; proximal tubules of the kidney; the adrenal medulla; and syncytiotrophoblasts and decidua cells of the placenta. GPER was also expressed in hepatocellular, pancreatic, renal, and endometrial cancers, pancreatic neuroendocrine tumours, and pheochromocytomas. The novel antibody 20H15L21 will serve as a valuable tool for basic research and the identification of GPER-expressing tumours during histopathological examinations.

Reassessment of SST4 Somatostatin Receptor Expression Using SST4-eGFP Knockin Mice and the Novel Rabbit Monoclonal Anti-Human SST4 Antibody 7H49L61.

Among the five somatostatin receptors (SST1-SST5), SST4 is the least characterized, which is in part due to the lack of specific monoclonal antibodies. We generated a knockin mouse model that expresses a carboxyl-terminal SST4-eGFP fusion protein. In addition, we extensively characterized the novel rabbit monoclonal anti-human SST4 antibody 7H49L61 using transfected cells and receptor-expressing tissues. 7H49L61 was then subjected to immunohistochemical staining of a series of formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Characterization of SST4-eGFP mice revealed prominent SST4 expression in cortical pyramidal cells and trigeminal ganglion cells. In the human cortex, 7H49L61 disclosed a virtually identical staining pattern. Specificity of 7H49L61 was demonstrated by detection of a broad band migrating at 50-60 kDa in immunoblots. Tissue immunostaining was abolished by preadsorption of 7H49L61 with its immunizing peptide. In the subsequent immunohistochemical study, 7H49L61 yielded a predominant plasma membrane staining in adrenal cortex, exocrine pancreas, and placenta. SST4 was also found in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. Altogether, we provide the first unequivocal localization of SST4 in normal and neoplastic human tissues. The monoclonal antibody 7H49L61 may also prove of great value for identifying SST4-expressing tumors during routine histopathological examinations.

Immunohistochemical Evaluation of Adaptor Protein FAM159B Expression in Normal and Neoplastic Human Tissues.

FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B's predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples.

Gender-Specific Efficacy Revealed by Head-to-Head Comparison of Pasireotide and Octreotide in a Representative In Vivo Model of Nonfunctioning Pituitary Tumors.

Invasive nonfunctioning pituitary tumors (NFPTs) are non-resectable neoplasms associated with frequent relapse and significant comorbidities. Current treatments, including somatostatin receptor 2 (SSTR2)-directed somatostatin analogs (SSAs), often fail against NFPTs. Thus, identifying effective therapies is clinically relevant. As NFPTs express SSTR3 at high levels, pasireotide, a multireceptor-targeted SSA, might be beneficial. Here we evaluated pasireotide in the only representative model of spontaneous NFPTs (MENX rats) in vivo. Octreotide long-acting release (LAR), pasireotide LAR, or placebo, were administered to age-matched, tumor-bearing MENX rats of both sexes for 28 d or 56 d. Longitudinal high-resolution magnetic resonance imaging monitored tumor growth. While tumors in placebo-treated rats increased in volume over time, PTs in drug-treated rats displayed significant growth suppression, and occasional tumor shrinkage. Pasireotide elicited stronger growth inhibition. Radiological responses correlated with tumors' proliferation rates. Both SSAs, but especially pasireotide, were more effective in female vs. male rats. Basal Sstr3 expression was significantly higher in the former group. It is noteworthy that female human NFPTs patients also have a trend towards higher SSTR3 expression. Altogether, our studies provide the rationale for testing pasireotide in patients with residual/recurrent NFPTs. If confirmed, the sex-related SSTR3 expression might be used as criteria to stratify NFPTs patients for treatment with pasireotide.

Immunohistochemical identification of complement peptide C5a receptor 1 (C5aR1) in non-neoplastic and neoplastic human tissues.

The complement component C5a and its receptor C5aR1 are involved in the development of numerous inflammatory diseases. In addition to immune cells, C5aR1 is expressed in neoplastic cells of multiple tumour entities, where C5aR1 is associated with a higher proliferation rate, advanced tumour stage, and poor patient outcomes. The aim of the present study was to obtain a broad expression profile of C5aR1 in human non-neoplastic and neoplastic tissues, especially in tumour entities not investigated in this respect so far. For this purpose, we generated a novel polyclonal rabbit antibody, {5227}, against the carboxy-terminal tail of C5aR1. The antibody was initially characterised in Western blot analyses and immunocytochemistry using transfected human embryonic kidney (HEK) 293 cells. It was then applied to a large series of formalin-fixed, paraffin-embedded non-neoplastic and neoplastic human tissue samples. C5aR1 was strongly expressed by different types of immune cells in the majority of tissue samples investigated. C5aR1 was also present in alveolar macrophages, bronchial, gut, and bile duct epithelia, Kupffer cells, occasionally in hepatocytes, proximal renal tubule cells, placental syncytiotrophoblasts, and distinct stem cell populations of bone marrow. C5aR1 was also highly expressed in the vast majority of the 32 tumour entities investigated, where a hitherto unappreciated high prevalence of the receptor was detected in thyroid carcinomas, small-cell lung cancer, gastrointestinal stromal tumours, and endometrial carcinomas. In addition to confirming published findings, we found noticeable C5aR1 expression in many tumour entities for the first time. Here, it may serve as an interesting target for future therapies.

Prognostic value of PD-L1 expression in bronchopulmonary neuroendocrine tumours.

Programmed death protein 1 (PD-1) and its ligand, PD-L1, have emerged as promising therapeutic targets for many types of cancer that overexpress PD-L1. However, data on PD-L1 expression levels in bronchopulmonary neuroendocrine neoplasms (BP-NEN) are limited and contradictory. In the present study, a total of 298 archived, formalin-fixed, paraffin-embedded BP-NEN samples from 97 patients diagnosed with typical carcinoid (TC), atypical carcinoid (AC), small cell lung cancer (SCLC), or large cell neuroendocrine carcinoma of the lung (LCNEC) were evaluated for PD-L1 expression by immunohistochemistry using the highly sensitive monoclonal anti-PD-L1 antibody 73-10. PD-L1 expression levels were semiquantitatively estimated by tumour grading. Of the 298 BP-NEN samples, 85% were positive for PD-L1 expression. PD-L1 immunostaining predominantly localized to the plasma membrane of both tumour cells and tumour-infiltrating immune cells. SCLC and LCNEC exhibited significantly higher PD-L1 expression levels than TC or AC. PD-L1 expression levels were also higher in patients with lymph node or distant metastases, in patients who smoked, and in patients who died during the follow-up period. Moreover, PD-L1 expression levels correlated positively with tumour grading, Ki-67 index and the expression of the chemokine receptor CXCR4 and negatively with the levels of somatostatin receptor 1 and chromogranin A. High tumour PD-L1 levels were associated with poor patient outcomes. In conclusion, PD-L1 expression is common in BP-NEN, increases with malignancy, and is associated with poor prognosis. Therefore, targeting the PD-1/PD-L1 axis could be a promising strategy for treating BP-NEN. PD-L1 may also represent a useful prognostic biomarker for this tumour entity.

Targeting Complement C5a Receptor 1 for the Treatment of Immunosuppression in Sepsis.

Complement factor C5a was originally identified as a powerful promoter of inflammation through activation of the C5a receptor 1 (C5ar1). Recent evidence suggests involvement of C5a not only in pro- but also in anti-inflammatory signaling. The present study aims to unveil the role of C5ar1 as potential therapeutic target in a murine sepsis model. Our study discloses a significantly increased survival in models of mild to moderate but not severe sepsis of C5ar1-deficient mice. The decreased mortality of C5ar1-deficient mice is accompanied by improved pathogen clearance and largely preserved liver function. C5ar1-deficient mice exhibited a significantly increased production of the pro-inflammatory mediator interferon-γ (IFN-γ) and a decreased production of the anti-inflammatory cytokine interleukin-10 (IL-10). Together, these data uncover C5a signaling as a mediator of immunosuppressive processes during sepsis and describe the C5ar1 and related changes of the IFN-γ to IL-10 ratio as markers for the immunological (dys)function accompanying sepsis.

Evaluation of Somatostatin and CXCR4 Receptor Expression in a Large Set of Prostate Cancer Samples Using Tissue Microarrays and Well-Characterized Monoclonal Antibodies.

BACKGROUND: Prostate cancer (PCa) is the most common type of cancer among men in Western countries. Despite numerous therapeutic options, few treatments are available for patients with end-stage disease. In the present study, different somatostatin receptors (SSTs) and the chemokine receptor CXCR4 were evaluated for their suitability as novel therapeutic targets in PCa. MATERIALS AND METHODS: The expression of SST subtypes 1, 2A, 3, and 5 and of CXCR4 was evaluated in 276 PCa tumor samples on a tissue microarray (TMA) in 23 whole-block tumor samples and in 3 PCa cell lines by immunohistochemistry using well-characterized monoclonal antibodies. RESULTS: Overall, the frequency and intensity of expression of SSTs and CXCR4 were very low among the PCa samples investigated. Specifically, SST5, SST2A, and SST3 were expressed, albeit at low intensity, in 10.5%, 9.1%, and 0.7% of the TMA samples, respectively. None of the TMA samples showed SST1 or CXCR4 expression. Only a single small-cell-type neuroendocrine carcinoma that was coincidentally included among the whole-block samples exhibited strong SST2A, SST5, and CXCR4 and moderate SST3 expression. Independent of the tumor cells, the tumor capillaries in many of the PCa samples were strongly positive for SST2A, SST3, SST5, or CXCR4 expression. SST expression in the tumor cells was associated with advanced tumor grade and stage. CONCLUSION: Overall, SST and CXCR4 expression levels are clearly of no therapeutic relevance in PCa. SST- or CXCR4-based therapy might be feasible, however, in rare cases of small-cell-type neuroendocrine carcinoma of the prostate.

Case report of a cystic parathyroidal adenoma with rapid growth induced by cinacalcet.

BACKGROUND: Primary hyperparathyroidism is a rare condition of disease which can seldomly present as giant retrotrhyroideal cysts, complicating the localization of the adenoma to resect. CASE PRESENTATION: A 56-year old female presented with hypercalcaemia of 3.38 mmol/L (2.2-2.65 mmol/L) and a history of breast cancer. A fast growing cystic parathyroidal adenoma was diagnosed by a multimodal approach including comprehensive diagnostic imaging (ultrasonography, scintigraphies, dynamic MRI) and cytopathological investigations after ultrasonography-guided puncture. The patient was cured by surgical extraction of the whole adenoma. In retrospect, the rapid growth was most likely induced by cinacalcet (initially 30 mg/d, later 60 mg/d) therapy which the patient received for few months only. Primary hyperparathyroidism was ascertained because surgical removal of the solitary adenoma cured the patient. Furthermore, there was no relevant renal insufficiency or history of prolonged calcium-level deregulation. CONCLUSIONS: This phenomenon of cystic degeneration of parathyroidal adenoma under therapy with cinacalcet has previously been described in secondary hyperparathyroidism, but not in primary hyperparathyroidism and should be considered in diagnostic approach.

Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke.

Monocyte-derived and tissue-resident macrophages are ontogenetically distinct components of the innate immune system. Assessment of their respective functions in pathology is complicated by changes to the macrophage phenotype during inflammation. Here we find that Cxcr4-CreER enables permanent genetic labeling of hematopoietic stem cells (HSCs) and distinguishes HSC-derived monocytes from microglia and other tissue-resident macrophages. By combining Cxcr4-CreER-mediated lineage tracing with Cxcr4 inhibition or conditional Cxcr4 ablation in photothrombotic stroke, we find that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue. After transient focal ischemia, Cxcr4 deficiency reduces monocyte infiltration and blunts the expression of pattern recognition and defense response genes in monocyte-derived macrophages. This is associated with an altered microglial response and deteriorated outcomes. Thus, Cxcr4 is essential for an innate-immune-system-mediated defense response after cerebral ischemia. We further propose Cxcr4-CreER as a universal tool to study functions of HSC-derived cells.