Heikki Huhta

Toll-like receptors 1, 2, 4 and 6 in esophageal epithelium, Barrett's esophagus, dysplasia and adenocarcinoma

Background Toll-like receptors (TLRs) recognize microbial and endogenous ligands and have already shown to play a role in esophageal cancer. In this study, we evaluated especially TLRs that sense bacterial cell wall components in Barretts esophagus, dysplasia and esophageal adenocarcinoma. Methods TLRs 1, 2, 4 and 6 were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal dysplasia (n = 30) or adenocarcinoma (n = 99). Structures and lesions were evaluated including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37), and esophageal adenocarcinoma (n = 99). Results We found TLR1, TLR2, TLR4 and TLR6 expression in all lesions. TLR expression increased in Barretts mucosa and dysplasia. There was profound increase of TLR expression from gastric- to intestinal-type columnar epithelium. In cancers, high nuclear and cytoplasmic staining of TLR4 associated with metastatic disease and poor prognosis. Conclusions TLR1, TLR2, TLR4 and TLR6 are upregulated during malignant changes of esophageal columnar epithelium. Increased TLR4 expression associates with advanced stage and poor prognosis in esophageal adenocarcinoma.

The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

Toll-like receptors (TLRs) are innate immune receptors expressed in all parts of the alimentary tract. However, analyses comparing expression in different segments and data on germ-free animals are lacking. Alimentary tract cancers show increased TLR expression. According to the field effect concept, carcinogenetic factors induce subtle cancer predisposing alterations in the whole organ. We studied TLR1 to TLR9 expression in all segments of the alimentary tract from cancer patients’ tumor-adjacent normal mucosa, healthy organ donors, and conventional and germ-free mice by using immunohistochemistry and quantitative PCR. All TLRs were expressed in all segments of the alimentary tract. Expression was most intensive in the small intestine in humans and conventional mice, but germ-free mice showed less expression in the small intestine. TLR expression levels were similar in cancer patients and organ donors. We provide systematic baseline data on the TLR expression in the alimentary tract. Normal epithelium adjacent to tumor seems to have similar TLR expression compared with healthy tissues suggesting absence of any field effect in TLR expression. Accordingly, specimens from cancer patients’ normal tumor-adjacent tissue can be used as control tissues in immunohistochemical TLR studies in gastrointestinal cancer.

Toll-Like Receptor 4 Wild Type Homozygozity of Polymorphisms +896 and +1196 Is Associated with High Gastrin Serum Levels and Peptic Ulcer Risk

Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

Nucleic acid-sensing toll-like receptors 3, 7 and 8 in esophageal epithelium, barrett’s esophagus, dysplasia and adenocarcinoma

ABSTRACT Toll-like receptors (TLRs) are immunological receptors recognizing various microbial and endogenous ligands, such as DNA, RNA, and other microbial and host components thus activating immunological responses. The expression of TLRs in esophageal adenocarcinoma is not well known. The aim of this study was to evaluate expression patterns of those TLRs that sense nucleic acids in Barrett’s esophagus with and without dysplasia and in esophageal adenocarcinoma. TLRs 3, 7 and 8 were stained immunohistochemically and evaluated in a cohort of patients with esophageal adenocarcinoma or dysplasia. Specimens with normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were studied. We used immunofluorescence to confirm the subcellular localization of TLRs. We found abundant expression of TLR3, 7 and 8 in esophageal squamous epithelium, columnar metaplasia, dysplasia and adenocarcinoma. Cytoplasmic expression of TLR3, TLR7 or TLR8 did not associate to clinicopathological parameters or prognosis in esophageal cancer. High nuclear expression of TLR8, confirmed with immunofluorescence, in cancer cells was observed in tumors of high T-stage (p < 0.01) and in tumors with organ metastasis (p < 0.001). High nuclear TLR8 expression was associated with poor prognosis (p < 0.001). The expression of TLR3, TLR7 and TLR8 increased toward dysplasia and adenocarcinoma. We demonstrated nuclear localization of TLR8, which associates with metastasis and poor prognosis. TLR3 and TLR7 do not seem to have prognostic significance in esophageal adenocarcinoma.

High toll-like receptor (TLR) 9 expression is associated with better prognosis in surgically treated pancreatic cancer patients

Pancreatic cancer remains one of the deadliest malignancies in the world. Inflammatory response and tumor environment are thought to play a major role in its pathogenesis. Knowledge on TLR expression and impact on patient survival in pancreatic cancer is limited. The study’s aim was to clarify the role of different TLRs in pancreatic cancer. TLR2, TLR4, and TLR9 expression was investigated in 65 surgically resected pancreatic ductal adenocarcinoma specimens by immunohistochemistry. The association between TLR expression, clinical parameters, and local inflammatory response to the tumor was assessed using chi-square test. Relation between patient survival and TLR expression was calculated with multivariable Cox regression, adjusted for age, sex, and tumor stage. We found TLR2, TLR4, and TLR9 to be expressed in pancreatic cancer. There was no association between TLR expression and tumor stage, tumor size, lymph node metastasis, or tumor necrosis. Contrary to our initial hypothesis, high cytoplasmic TLR9 expression was associated with longer patient survival, and multivariate analysis identified low TLR9 expression as an independent risk factor for cancer-specific death (HR 3.090, 95% CI 1.673–5.706). The results suggest that high TLR9 expression in pancreatic ductal adenocarcinoma indicates improved prognosis. The prognostic effect of TLR9 might be associated with bacterial exposure, but this needs further evidence.

Intratumoral lactate metabolism in Barrett’s esophagus and adenocarcinoma

Background Monocarboxylate transporters (MCTs) are cell membrane proteins which transport pyruvate, lactate and ketone bodies across the plasma membrane. MCTs are activated in various cancers, but their expression in esophageal adenocarcinoma is not known. The present study was conducted to elucidate the expression of MCTs in esophageal adenocarcinoma and its precursor lesions. Results Cytoplasmic MCT1, MCT4 and MTCO1 expression linearly increased from normal epithelium to Barretts mucosa to dysplasia and cancer. Low cytoplasmic MCT1 expression associated with high T-class (P < 0.01), positive lymph node metastases (P < 0.05), positive distant metastases (P < 0.01) and high tumor stage (P < 0.01). High cytoplasmic MCT4 expression correlated significantly with positive distant metastases (P < 0.05). Both low MCT1 and high MCT4 histoscore predicted survival in univariate analysis (P < 0.01). MCT4 histoscore predicted survival in multivariate analysis (P = 0.043; HR 1.8 95%CI 1.0–3.1). MTCO1 expression was not correlated to clinicopathological variables or survival. Materials and Methods MCT1, MCT4 and mitochondrial cytochrome c oxidase (MTCO1) expression were determined with immunohistochemistry in esophageal specimens from 129 patients with columnar dysplasia or adenocarcinoma. Specimens including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51), low-grade (n = 42), high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were evaluated. Conclusions Major increase in markers of tumor metabolism occurs during carcinogenesis and progression of esophageal adenocarcinoma. MCT1 and MCT4 are prognostic factors in esophageal adenocarcinoma.

Localization of nucleic acid-sensing toll-like receptors in human and mouse pancreas.

Nucleic acid‐sensing toll‐like receptors (TLRs) (3, 7, 8, 9) have a role both in antiviral innate immunity and in autoimmune disorders. We assessed the expression of TLR3, 7, 8 and 9 in human and mouse pancreas focusing on the subpopulations of cells in the Langerhans islets. We studied eight human samples with normal pancreatic islets and two samples from patients with type 1 diabetes. Additionally, 10 CD‐1 mouse pancreases were analysed. Immunohistochemical double‐stainings for the TLRs and insulin, glucagon or somatostatin, respectively, were performed along with appropriate controls. In human pancreas, strong immunoreaction of TLR7 and TLR8 was observed in the insulin‐positive beta cells, whereas glucagon‐ or somatostatin‐expressing cells of the islets were weakly stained or negative. In type 1 diabetes, the expression in islets was weak or lost (TLR7: p = 0.014, TLR8: p = 0.053), correlating with loss of beta cells. TLR3 and 9 were expressed only weakly with no correlation with specific cell types. In mouse pancreas, only TLR9 was detected. Intra‐pancreatic nerve ganglia strongly expressed TLR7. The strong expression of TLR7 and TLR8 in the beta cells of normal human islets could be an important piece in the puzzle of type 1 diabetes pathogenesis, and be linked with destruction of this particular subpopulation of the islet cells. In normal mice, only TLR9 can be constantly detected in the islets, highlighting differences between the species.

Weak HIF-1alpha expression indicates poor prognosis in resectable pancreatic ductal adenocarcinoma

BackgroundHIF-1alpha and CAIX proteins are commonly expressed under hypoxic conditions, but other regulatory factors have been described as well. Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and strong stromal reaction and has a dismal prognosis with the currently available treatment modalities.MethodsWe investigated the expression and prognostic role of HIF-1alpha and CAIX in PDAC series from Northern Finland (n = 69) using immunohistochemistry.ResultsIn our PDAC cases, 95 and 85% showed HIF-1alpha and CAIX expression, respectively. Low HIF-1alpha expression correlated with poor prognosis, and multivariate analysis identified weak HIF-1alpha intensity as an independent prognostic factor for PDAC-specific deaths (HR 2.176, 95% CI 1.216–3.893; p = 0.009). There was no correlation between HIF-1alpha and CAIX expression levels, and the latter did not relate with survival.ConclusionsOur findings are in contrast with previous research by finding an association between low HIF-1alpha and poor prognosis. The biological mechanisms remain speculative, but such an unexpected relation with prognosis and absence of correlation between HIF-1alpha and CAIX suggests that the prognostic association of HIF-1alpha may not directly be linked with hypoxia. Accordingly, the role of HIF-1alpha might be more complex than previously thought and the use of this marker as a hypoxia-related prognostic factor should be addressed with caution.

Carbonic anhydrases II, IX, and XII in Barrett’s esophagus and adenocarcinoma

The aim of our retrospective study was to investigate the expression and clinical significance of the cancer-associated carbonic anhydrases (CAs) II, IX, and XII in Barrett’s esophagus and esophageal adenocarcinoma (EAC). We evaluated 101 archival specimens from patients with EAC as well as seven and 26 samples from patients with high-and low-grade dysplasia, respectively. In addition, normal esophageal squamous epithelium, gastric, and intestinal metaplasia were analyzed when present. The expression patterns of isozymes were detected by immunohistochemistry. CAII and CIX expression levels were lower in the squamous epithelium than in the columnar cells while CAXII showed an opposite pattern and was present mainly in squamous epithelium. Expression patterns in benign, dysplastic, or malignant esophageal columnar lesions were not significantly different. The assessment of clinicopathological associations showed that CAII was significantly downregulated in metastatic disease (p = 0.026). CAIX showed no association with prognosis, although there appeared to be an association (p = 0.056) between high expression and nodal spread. In conclusion, CAII, CAIX, and CAXII do not serve as biomarkers for different phases in the development of EAC.

Effect of Sildenafil on Pulmonary Circulation and Cardiovascular Function in Near-Term Fetal Sheep During Hypoxemia

Sildenafil is a potential new treatment for placental insufficiency in human pregnancies as it reduces the breakdown of vasodilator nitric oxide. Pulmonary vasodilatation is observed in normoxemic fetuses following sildenafil administration. Placental insufficiency often leads to fetal hypoxemia that can cause pulmonary vasoconstriction and fetal cardiac dysfunction as evidenced by reduced isovolumic myocardial velocities. We tested the hypotheses that sildenafil, when given directly to the hypoxemic fetus, reverses reactive pulmonary vasoconstriction, increases left ventricular cardiac output by increasing pulmonary venous return, and ameliorates hypoxemic myocardial dysfunction. We used an instrumented sheep model. Fetuses were made hypoxemic over a mean (standard deviation) duration of 41.3 (9.5) minutes and then given intravenous sildenafil or saline infusion. Volume blood flow through ductus arteriosus was measured with an ultrasonic transit-time flow probe. Fetal left and right ventricular outputs and lung volume blood flow were calculated, and ventricular function was examined using echocardiography. Lung volume blood flow decreased and the ductus arteriosus volume blood flow increased with hypoxemia. There was a significant reduction in left ventricular and combined cardiac outputs during hypoxemia in both groups. Hypoxemia led to a reduction in myocardial isovolumic velocities, increased ductus venosus pulsatility, and reduced left ventricular myocardial deformation. Direct administration of sildenafil to hypoxemic fetus did not reverse the redistribution of cardiac output. Furthermore, fetal cardiac systolic and diastolic dysfunction was observed during hypoxemia, which was not improved by fetal sildenafil treatment. In conclusion, sildenafil did not improve pulmonary blood flow or cardiac function in hypoxemic sheep fetuses.