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Supplementary Figure 1 from Effect of Orally Administered Bovine Lactoferrin on the Growth of Adenomatous Colorectal Polyps in a Randomized, Placebo-Controlled Clinical Trial

Supplementary Methods, Table 1, Figure Legends 1-2 from Effect of Orally Administered Bovine Lactoferrin on the Growth of Adenomatous Colorectal Polyps in a Randomized, Placebo-Controlled Clinical Trial

Supplementary Figure 2 from Effect of Orally Administered Bovine Lactoferrin on the Growth of Adenomatous Colorectal Polyps in a Randomized, Placebo-Controlled Clinical Trial

Background Multi-walled carbon nanotubes can be divided into two general subtypes: tangled and straight. MWCNT-N (60 nm in diameter) and MWCNT-7 (80–90 nm in diameter) are straight-type MWCNTs, and similarly to asbestos, both are carcinogenic to the lung and pleura when administered to rats via the airway. Injection of straight-type MWCNTs into the peritoneal cavity also induces the development of mesothelioma, however, injection of tangled-type MWCNTs into the peritoneal cavity does not induce carcinogenesis. To investigate these effects in the lung we conducted a 2-year comparative study of the potential carcinogenicities of a straight-type MWCNT, MWCNT-A (approximately 150 nm in diameter), and a tangled-type MWCNT, MWCNT-B (7.4 nm in diameter) after administration into the rat lung. Crocidolite asbestos was used as the reference material, and rats administered vehicle were used as the controls. Test materials were administered by intra-Tracheal Intra-Pulmonary Spraying (TIPS) once a week over a 7 week period (8 administrations from day 1 to day 50), followed by a 2-year observation period without further treatment. Rats were administered total doses of 0.5 or 1.0 mg MWCNT-A and MWCNT-B or 1.0 mg asbestos. Results There was no difference in survival between any of the groups. The rats administered MWCNT-A or asbestos did not have a significant increase in bronchiolo-alveolar hyperplasia or tumors in the lung. However, the rats administered MWCNT-B did have significantly elevated incidences of bronchiolo-alveolar hyperplasia and tumors in the lung: the incidence of bronchiolo-alveolar hyperplasia was 0/20, 6/20, and 9/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively, and the incidence of adenoma and adenocarcinoma combined was 1/19, 5/20, and 7/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively. Malignant pleural mesothelioma was not induced in any of the groups. Conclusions The results of this initial study indicate that tangled-type MWCNT-B is carcinogenic to the rat lung when administered via the airway, and that straight-type MWCNT-A did not have higher carcinogenic potential in the rat lung than tangled-type MWCNT-B.

Background Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. Methods Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. Results DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p Conclusions Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

Alveolar macrophages are responsible for clearance of airborne dust and pathogens. How they recognize and phagocytose a variety of engineered nanomaterials (ENMs) with different properties is an important issue for safety assessment of ENMs. Surfactant-associated proteins, specifically existing in the pulmonary surfactant, are important opsonins for phagocytosis of airborne microorganisms. The purposes of the current study are to understand whether opsonization of ENMs by surfactant-associated proteins promotes phagocytosis of ENMs and cytokine production, and to determine whether a common pathway for phagocytosis of ENMs with different properties exists. For these purposes, four ENMs, MWCNT-7, TiO2, SiO2, and fullerene C60, with different shapes, sizes, chemical compositions, and surface reactivities, were chosen for this study. Short-term pulmonary exposure to MWCNT-7, TiO2, SiO2, and C60 induced inflammation in the rat lung, and most of the administered ENMs were phagocytosed by alveolar macrophages. The ENMs were phagocytosed by isolated primary alveolar macrophages (PAMs) in vitro, and phagocytosis was enhanced by rat bronchioalveolar lavage fluid (BALF), suggesting that proteins in the BALF were associated with phagocytosis. Analysis of proteins bound to the 4 ENMs by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Both BALF and SP-A, but not SP-D, enhanced TNF-α production by MWCNT-7 treated PAMs; BALF, SP-A, and SP-D increased IL-1β production in TiO2 and SiO2 treated PAMs; and BALF, SP-A, and SP-D enhanced IL-6 production in C60 treated PAMs. Knockdown of CD14, a receptor for SP-A/D, significantly reduced phagocytosis of ENMs and SP-A-enhanced cytokine production by PAMs. These results indicate that SP-A/D can opsonize all the test ENMs and enhance phagocytosis of the ENMs by alveolar macrophages through CD14, suggesting that SP-A/D-CD14 is a common pathway mediating phagocytosis of ENMs. Cytokine production induced by ENMs, however, is dependent on the type of ENM that is phagocytosed. Our results demonstrate a dual role for surfactant proteins as opsonins for both microbes and for inhaled dusts and fibers, including ENMs, allowing macrophages to recognize and remove the vast majority of these particles, thereby, greatly lessening their toxicity in the lung.

Carbon nanotubes (CNTs) are recently developed tubular nanomaterials, with diameters ranging from a few nanometers to tens of nanometers, and the length reaching up to several micrometers. They can be either single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs). Due to their nano-scaled structure, CNTs have a unique set of mechanical, electrical, and chemical properties that make them useful in information technologies, optoelectronics, energy technologies, material sciences, medical technologies, and other fields. However, with the wide application and increasing production of CNTs, their potential risks have led to concerns regarding their impact on environment and health. The shape of some types of CNTs is similar to asbestos fibers, which suggests that these CNTs may cause characteristic pleural diseases similar to those found in asbestos-exposed humans, such as pleural plaques and malignant mesothelioma. Experimental data indicate that CNTs can induce lung and pleural lesions, inflammation, pleural fibrosis, lung tumors, and malignant mesothelioma upon inhalation in the experimental animals. In this review, we focus on the potential of MWCNTs to induce diseases similar to those by asbestos, molecular and cellular mechanisms associated with these diseases, and we discuss a method for evaluating the pleural toxicity of MWCNTs.

Background Malignant pleural mesothelioma (MPM) is a debilitating disease of the pleural cavity. It is primarily associated with previous inhalation of asbestos fibers. These fibers initiate an oxidant coupled inflammatory response. Repeated exposure to asbestos fibers results in a prolonged inflammatory response and cycles of tissue damage and repair. The inflammation-associated cycles of tissue damage and repair are intimately involved in the development of asbestos-associated cancers. Macrophages are a key component of asbestos-associated inflammation and play essential roles in the etiology of a variety of cancers. Macrophages are also a source of C-C motif chemokine ligand 2 (CCL2), and a variety of tumor-types express CCL2. High levels of CCL2 are present in the pleural effusions of mesothelioma patients, however, CCL2 has not been examined in the serum of mesothelioma patients. Methods The present study was carried out with 50 MPM patients and 356 subjects who were possibly exposed to asbestos but did not have disease symptoms and 41 healthy volunteers without a history of exposure to asbestos. The levels of CCL2 in the serum of the study participants was determined using ELISA. Results Levels of CCL2 were significantly elevated in the serum of patients with advanced MPM. Conclusions Our findings are consistent with the premise that the CCL2/CCR2 axis and myeloid-derived cells play an important role in MPM and disease progression. Therapies are being developed that target CCL2/CCR2 and tumor resident myeloid cells, and clinical trials are being pursued that use these therapies as part of the treatment regimen. The results of trials with patients with a similar serum CCL2 pattern as MPM patients will have important implications for the treatment of MPM.

Multi‐walled carbon nanotube‐7 (MWCNT‐7) fibers are biopersistent and have a structure similar to asbestos. MWCNT‐7 has been shown to induce malignant mesothelioma when administered by intrascrotal or intraperitoneal injection in rats and mice, and an inhalation study demonstrated that rats exposed to respirable MWCNT‐7 developed lung tumors. MWCNT‐N, which is similar to MWCNT‐7, was shown to induce both lung tumors and malignant mesothelioma in rats when administered by trans‐tracheal intrapulmonary spraying (TIPS). The present study was performed to investigate the carcinogenicity of MWCNT‐7 when administered by the TIPS method. Ten‐week‐old male F344/Crj rats were divided into 3 groups and administered 0.5 mL vehicle, 0.250 μg/mL MWCNT‐7 or 0.250 μg/mL crocidolite once a week for 12 weeks (total doses of 1.5 mg/rat) and then observed for up to 104 weeks. Rats in the MWCNT‐7 group began to die from pathologies associated with the development of malignant mesothelioma 35 weeks after the final TIPS administration. Overall, the incidence of malignant mesothelioma in the MWCNT‐7 group was significantly higher than in the vehicle or crocidolite groups.

Background: Alveolar macrophages are responsible for clearance of airborne dust and pathogens. How they recognize and phagocytose a variety of engineered nanomaterials (ENMs) with different properties is an important issue for safety assessment of ENMs. Surfactant-associated proteins, specifically existing in the pulmonary surfactant, are important opsonins for phagocytosis of airborne microorganisms. The purposes of the current study are to understand whether opsonization of ENMs by surfactant-associated proteins promotes phagocytosis of ENMs and cytokine production and to find out a common pathway for ENMs with different properties. Results: 4 ENMs including MWCNT-7, TIO2, SIO2, and fullerene C60, each with different shape, size, chemical composition and surface reactivity, were chosen for the study. Short-term pulmonary exposure of MWCNT-7, TIO2, SIO2, and C60 induced inflammation in the rat lung, and most of the administered ENMs were phagocytosed by alveolar macrophages. The ENMs were phagocytosed by isolated primary alveolar macrophages (PAMs) in vitro, which was enhanced by the rat bronchioalveolar lavage fluid (BALF), suggesting that proteins in BALF were associated with the phagocytosis. Further analysis of the 4 ENMs-bound proteins by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Like BALF, SP-A, but not SP-D, enhanced TNF-a production in the MWCNT-7-treated PAMs; both SP-A and SP-D increased IL-b production in the TIO2-or SIO2-stimulated PAMs; while SP-A and SP-D enhanced IL-6 production in the C60-stimulated PAMs. Knockdown of CD14, a receptor for SP-A/D, significantly reduced the cytokine production and ENMs phagocytosis. Conclusions: These results indicate that SP-A/D can opsonize all the studied ENMs to enhance phagocytosis of the ENMs by alveolar macrophages through CD14, suggesting that SP-A/D-CD14 is a common pathway mediating phagocytosis of ENMs. Cytokine production induced by ENMs, however, is dependent on what an ENM is phagocytosed. Our results are helpful for the understanding of clearance of ENMs by alveolar macrophages and mechanisms of different ENMs-induced lung toxicity.

Patients with ulcerative colitis or colonic Crohn’s disease have a significantly increased risk of developing colorectal cancer. Bovine lactoferrin (bLF) reportedly inhibited the development of colon cancer in rats and mice, and in a placebo controlled trial, ingestion of bLF inhibited the growth of intestinal polyps. In addition, in a case study, a patient with Crohn’s disease was reported to have remained in remission for over 7 years while ingesting 1 g of bLF daily. Thus, bLF has an inhibitory effect on colon carcinogenesis, and it may also promote remission of Crohn’s disease. The purpose of this study was to investigate the effects of bLF in a mouse model of colorectal cancer related to irritable bowel disease (IBD). The mice were divided into 4 groups: (i) no treatment; (ii) treated with bLF only; (iii) treated with azoxymethane plus dextran sulfate sodium (AOM + DSS); and (iv) treated with AOM + DSS + bLF. AOM was used to initiate intestinal cancer, and DSS was used to induce IBD-like inflammation in the intestine of the C57BL/6 mice. At the end of the study, the mice treated with AOM + DSS + bLF had a better fecal score, fewer lesions in the colon, and less weight loss than the mice treated with AOM + DSS without bLF. However, there were no statistically significant differences between the two groups with respect to tumor burden.

Potassium octatitanate (K2O·8TiO2, POT) fibers are used as an alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, inhalation studies have shown that respired POT fibers have little carcinogenic potential. We conducted a short-term study in which we administered POT fibers, and anatase and rutile titanium dioxide nanoparticles (a-nTiO2, r-nTiO2) to rats using intra-tracheal intra-pulmonary spraying (TIPS). We found that similarly to other materials, POT fibers were more toxic than non-fibrous nanoparticles of the same chemical composition, indicating that the titanium dioxide composition of POT fibers does not appear to account for their lack of carcinogenicity. The present report describes the results of the 3-week and 52-week interim killing of our current 2-year study of POT fibers, with MWCNT-7 as a positive control and r-nTiO2 as a non-fibrous titanium dioxide control. Male F344 rats were administered 0.5 ml vehicle, 62.5 µg/ml and 125 µg/ml r-nTiO2 and POT fibers, and 125 µg/ml MWCNT-7 by TIPS every other day for 2 weeks (eight doses: total doses of 0.25 and 0.50 mg/rat). At 1 year, POT and MWCNT-7 fibers induced significant increases in alveolar macrophage number, granulation tissue in the lung, bronchiolo-alveolar cell hyperplasia and thickening of the alveolar wall, and pulmonary 8-OHdG levels. The 0.5 mg POT- and the MWCNT-7-treated groups also had increased visceral and parietal pleura thickness, increased mesothelial cell PCNA labeling indices, and a few areas of visceral mesothelial cell hyperplasia. In contrast, in the r-nTiO2-treated groups, none of the measured parameters were different from the controls.

Many approaches have been taken to identify new biomarkers of pancreatic ductal carcinoma (PDC). Since animal models can be sampled under controlled conditions, better standardization is possible compared with heterogeneous human studies. Transgenic rats with conditional activation of oncogenic RAS in pancreatic tissue develop PDC that closely resembles the biological and histopathological features of human PDC. Using this model, we evaluated the usefulness of leucine-rich α2-glycoprotein-1 (LRG-1) as a serum marker. In this study, we found that LRG-1 was overexpressed in rat PDC compared with normal pancreas tissue of the control rats. Serum levels of LRG-1 were also significantly higher in rats bearing PDC than in controls. Importantly, chronic pancreatitis in male Wistar Bonn/Kobori rats, which is a widely accepted as a model of chronic pancreatitis, did not cause serum levels of LRG-1 to become elevated. These results strongly support serum LRG-1 as a candidate biomarker for noninvasive diagnosis of PDC. Our models of pancreas cancer provide a useful strategy for evaluation of candidate markers applicable to human cancer.

Background: CD73 is an ectonucleotidase regulating extracellular adenosine concentration and plays an important role in adenosine-mediated immunosuppressive pathways. The efficacy of CD73-targeted therapy depends on the expression levels of CD73, therefore, monitoring CD73 status in cancer patients would provide helpful information for selection of patients who would benefit from CD73-targeted therapy. Here, we evaluated the ability of 111In-labeled antibody 067-213, which has high affinity for human CD73, to act as a noninvasive imaging probe. Methods: Cell binding and competitive inhibition assays for 111In-labeled 067-213 were conducted using MIAPaCa-2 (high CD73 expression) and A431 (low CD73 expression) cells. For in vivo assessments, biodistribution and SPECT/CT studies were conducted in MIAPaCa-2 and A431 tumor-bearing mice. To estimate the absorbed dose in humans, biodistribution and SPECT/CT studies were conducted in healthy rats. Results: 111In-labeled 067-213 bound to MIAPaCa-2 and A431 cells in a CD73-dependent manner and the affinity loss after 111In-labeling was limited. Biodistribution and SPECT/CT studies with 111In-labeled 067-213 in mice showed high uptake in MIAPaCa-2 tumors and lower uptake in A431 tumors. In rats, the probe did not show high uptake in normal organs, including endogenously CD73-expressing organs. The estimated absorbed doses in humans were reasonably low. Conclusions: 111In-labeled 067-213 showed CD73-expression-dependent tumor uptake and low uptake in normal organs and tissues. Radiolabeled 067-213 holds promise as an imaging probe for noninvasive evaluation of CD73 expression levels in patients. Our data encourage further clinical studies to clarify a role for CD73 monitoring in patients receiving CD73-targeted immune therapy.

Very little is known about the in vivo toxicity of inhaled double-walled carbon nanotubes (DWCNTs). In the present study, we compared the pulmonary toxicity of DWCNT to MWCNT-7, a well-known multi-walled carbon nanotube. Rats were divided into six groups: untreated, vehicle, low-dose DWCNT, high-dose DWCNT, low-dose MWCNT-7, and high-dose MWCNT-7. The test materials were administered by intra-tracheal intra-pulmonary spraying (TIPS) every other day for 15 days: the low-dose and high-dose groups were administered final total doses of 0.25 and 0.50 mg/rat of the test material. The animals were sacrificed 1 and 6 weeks after the final TIPS administration. Six weeks after the final TIPS administration, rats administered MWCNT-7 had high levels of macrophage infiltration into the lung with dense alveolar wall fibrous thickening throughout the lung; significant elevation of lactate dehydrogenase activity, alkaline phosphatase activity, and total protein concentration in the bronchioalveolar lavage fluid; an increase in the pulmonary cell PCNA index; slightly elevated levels of 8-OHdG DNA adducts in lung tissue DNA; a small but significant increase in protein concentration in the pleural cavity lavage fluid and an increase in the visceral mesothelial cell PCNA index. None of these parameters was increased in rats administered DWCNT. The primary lesion in rats administered DWCNT was scattered formation of granulation tissue containing internalized DWCNT fibers. Our data indicate that DWCNT has lower pulmonary and pleural toxicity than MWCNT-7. Electronic supplementary material The online version of this article (10.1007/s00204-018-2336-3) contains supplementary material, which is available to authorized users.

Potassium octatitanate fibers (K2O·8TiO2, POT fibers) are widely used as an alternative to asbestos. We investigated the pulmonary and pleural toxicity of POT fibers with reference to 2 non‐fibrous titanium dioxide nanoparticles (nTiO 2), photoreactive anatase (a‐nTiO 2) and inert rutile (r‐nTiO 2). Ten‐week‐old male F344 rats were given 0.5 mL of 250 μg/mL suspensions of POT fibers, a‐nTiO 2, or r‐nTiO 2, 8 times (1 mg/rat) over a 15‐day period by trans‐tracheal intrapulmonary spraying (TIPS). Rats were killed at 6 hours and at 4 weeks after the last TIPS dose. Alveolar macrophages were significantly increased in all treatment groups at 6 hours and at 4 weeks. At week 4, a‐nTiO 2 and r‐nTiO 2 were largely cleared from the lung whereas a major fraction of POT fibers were not cleared. In the bronchoalveolar lavage, alkaline phosphatase activity was elevated in all treatment groups, and lactate dehydrogenase (LDH) activity was elevated in the a‐nTiO 2 and POT groups. In lung tissue, oxidative stress index and proliferating cell nuclear antigen (PCNA) index were elevated in the a‐nTiO 2 and POT groups, and there was a significant elevation in C‐C motif chemokine ligand 2 (CCL2) mRNA and protein in the POT group. In pleural cavity lavage, total protein was elevated in all 3 treatment groups, and LDH activity was elevated in the a‐nTiO 2 and POT groups. Importantly, the PCNA index of the visceral mesothelium was increased in the POT group. Overall, POT fibers had greater biopersistence, induced higher expression of CCL2, and provoked a stronger tissue response than a‐nTiO 2 or r‐nTiO 2.

Background Potassium octatitanate fibers (K2O•8TiO2, POT fibers) are used as an asbestos substitute. Their physical characteristics suggest that respirable POT fibers are likely to be carcinogenic in the lung and pleura. However, previous 2-year inhalation studies reported that respired POT fibers had little or no carcinogenic potential. In the present study ten-week old male F344 rats were left untreated or were administered vehicle, 0.25 or 0.5 mg rutile-type nano TiO2 (r-nTiO2), 0.25 or 0.5 mg POT fibers, or 0.5 mg MWCNT-7 by intra-tracheal intra-pulmonary spraying (TIPS), and then observed for 2 years. Results There were no differences between the r-nTiO2 and control groups. The incidence of bronchiolo-alveolar cell hyperplasia was significantly increased in the groups treated with 0.50 mg POT and 0.50 mg MWCNT-7. The overall incidence of lung tumors, however, was not increased in either the POT or MWCNT-7 treated groups. Notably, the carcinomas that developed in the POT and MWCNT-7 treated rats were accompanied by proliferative fibrous connective tissue while the carcinomas that developed in the untreated rats and the r-nTiO2 treated rats were not (carcinomas did not develop in the vehicle control rats). In addition, the carcinoma that developed in the rat treated with 0.25 mg POT was a squamous cell carcinoma, a tumor that develops spontaneously in about 1 per 1700 rats. The incidence of mesothelial cell hyperplasia was 4/17, 7/16, and 10/14 and the incidence of malignant mesothelioma was 3/17, 1/16, and 2/14 in the 0.25 mg POT, 0.5 mg POT, and MWCNT-7 treated groups, respectively. Neither mesothelial cell hyperplasia nor mesothelioma developed in control rats or the rats treated with r-nTiO2. Since the incidence of spontaneously occurring malignant mesothelioma in rats is extremely low, approximately 1 per 1000 animals (Japan Bioassay Research Center [JBRC] historical control data), the development of multiple malignant mesotheliomas in the POT and MWCNT-7 treated groups was biologically significant. Conclusion The incidence of pleural mesotheliomas in male F344 rats administered POT fibers and MWCNT-7 was significantly higher than the JBRC historical control data, indicating that the incidence of pleural mesothelioma in the groups administered POT fibers and MWCNT-7 fibers via the airway using TIPS was biologically significant. The incidence of type II epithelial cell hyperplasia and the histology of the carcinomas that developed in the POT treated rats also indicates that respirable POT fibers are highly likely to be carcinogenic in the lungs of male F344 rats. Electronic supplementary material The online version of this article (10.1186/s12989-019-0316-2) contains supplementary material, which is available to authorized users.

This paper examines lower bounds on direction-of-arrival (DOA) estimation performance for radar systems with considerations made for wideband mutual coupling. For typical half-wavelength element spacing, array elements exhibit a degree of interaction with each other that may alter the currents on each element. We account for mutual coupling in the wideband case by implementing a least-squares inverse solution for mutual coupling interactions as a function of frequency. A coupling-impacted received data covariance is produced, which allows us to observe the effect of coupling on performance lower bounds, including the Cramer-Rao Lower Bound (CRLB) and the Ziv-Zakai Bound (ZZB). The ZZB was the focus as it holds at low SNR. Using the aforementioned bounds, we examined the impact of mutual coupling on different array sizes and compared dense to sparse array architectures. Additionally, we compared chirp waveforms to band-limited noise to compare bounds for different waveform types. We found that mutual coupling has a greater impact on the lower bound for smaller array sizes, and a sparse array configuration was found most favorable. The two waveform types demonstrate very similar behavior.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT‐N (NIKKISO) after administration to the rat lung. MWCNT‐N was fractionated by passing it through a sieve with a pore size of 25 μm. The average lengths of the MWCNT were 4.2 μm before filtration and 2.6 μm in the flow‐through fraction; the length of the retained MWCNT could not be determined. For the present study, 10‐week‐old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT‐N before filtration, MWCNT‐N flow‐through and MWCNT‐N retained groups. Administration was by the trans‐tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo‐alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.

Carbon nanotubes (CNTs) are composed of one or more one-atom thick graphene cylinders. CNTs have remarkable mechanical, thermal, chemical, and electrical properties, and their tubular structure makes them very lightweight. However, these properties also make CNTs easily airborne and highly biopersistent, and respired CNTs have the potential to induce inflammatory lesions in tissues. Similar to asbestos, several MWCNTs were shown to induce peritoneal malignant mesotheliomas after intraperitoneal administration in rats and mice. MWCNT-N was the first CNT shown to induce lesions in lung and pleural tissue after administration via the airway: in this study, MWCNT-N was administered to rats using intratracheal, intrapulmonary spraying (TIPS). Shortly thereafter, MWCNT-7 was shown to induce bronchioloalveolar tumors in rats after inhalation exposure. These two MWCNTs have walls of more than 40 layers and form rod-shaped aggregates, and consequently have the potential to act similarly to asbestos and induce neoplastic lesions in the lung and/or pleural tissue. To investigate the effects of wall thickness on the carcinogenicity of MWCNTs, we conducted a 2-year study comparing 2 different types of MWCNTs, rigid rod-shaped MWCNT-A (213 layers, 150 nm in diameter, forming rigid rod shape aggregates) and flexible MWCNT-B (15-18 layers, 15 nm in diameter, forming tangled shaped aggregates). Crocidolite asbestos was used as the reference material. The rats were administered MWCNTs and asbestos using TIPS once a week over an 8-week period (total low and high doses of 0.5 and 1.0 mg/rat), followed by 2-year observation without further treatment (Suzui et al., 2016; Xu et al., 2014; Abdelgied and Elgazzar, 2019). In the MWCNT-B group, the combined incidence of bronchioloalveolar tumors was increased in the group administered 1.0 mg MWCNT-B compared to the vehicle control group. Malignant pleural mesothelioma was not induced in any of the groups. MWCNT-A and crocidolite asbestos also induced bronchioloalveolar tumors; however, the incidence was not statistically significant compared to the controls. The results indicate that the carcinogenic potential of inhaled flexible, tangled MWCNT-B is as high or higher than that of rigid, rod-shaped MWCNT-A. This is the first study to show that a thin, flexible MWCNT induces lung tumors after administration via the airway. Further studies are warranted to investigate the association of shape and rigidity with the carcinogenicity of respirable CNTs. Citation Format: Hiroyuki Tsuda, William T. Alexander, Takamasa Numano, Mohamed Ahmed Mahmoud Abdelgied, Ahmed Maher Mahmoud Ibrahim Elgazzar, Hiroshi Takase, Jiegou Xu, Aya Naiki, Satoru Takahashi, Akihiko Hirose, Makoto Ohnishi, Jun Kanno, David B. Alexander. Demonstration of the carcinogenicity of a flexible tangled multiwalled carbon nanotube in the rat lung [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A53.

The tumor suppressor protein RECK has been implicated in the regulation of matrix metalloproteinases (MMPs), NOTCH-signaling and WNT7-signaling. It remains unclear, however, how broad the spectrum of RECK targets extends. To find novel RECK binding partners, we took the unbiased approach of yeast two-hybrid screening. This approach detected ADAMTS10 as a RECK-interactor. ADAMTS10 has been characterized as a metalloproteinase involved in fibrillin-rich microfibril biogenesis, and its mutations have been implicated in the connective tissue disorder Weill-Marchesani syndrome. Experiments in vitro using recombinant proteins expressed in mammalian cells indicated that RECK indeed binds ADAMTS10 directly, that RECK protects ADAMTS10 from fragmentation following chemical activation and that ADAMTS10 interferes with the activity of RECK to inhibit MT1-MMP. In cultured cells, RECK increases the amount of ADAMTS10 associated with the cells. Hence, the present study has uncovered novel interactions between two molecules of known clinical importance, RECK and ADAMTS10. This article has an associated First Person interview with the first author of the paper., Summary: RECK plays critical roles in tumor suppression and embryogenesis, while its mechanisms of actions remain largely obscure. This study has revealed the novel interaction between RECK and ADAMTS10.

Translocation of multiwalled carbon nanotubes (MWCNTs) from the lung to the pleural cavity, deposition of the fibers in the pleural tissue, induction of pleural fibrosis, and mesothelial proliferation have been found in rodents administered MWCNTs by different pulmonary exposure methods. However, whether the translocation and deposition and the subsequent pleural inflammation are associated with the pleural lesions is unclear. In the present study, male F344 rats were given 250 μg of two types of MWCNTs, with crocidolite as a positive control, 2 times/week for 4 weeks by intratracheal spraying. At 24 h and at 3 months after the last spraying, the rats were sacrificed for histological examination of the lung and chest wall; pleural cavity lavage was also collected at sacrifice for observation of pleural inflammatory reactions. The results indicated that intratracheally sprayed MWCNTs, like crocidolite fibers, translocated into the pleural cavity, deposited in the pleura, and induced persistent infiltration of immune cells into the pleural cavity, persistent pleural fibrosis, and mesothelial proliferation. The number of MWCNT fibers detected in the pleural cavity lavage was parallel to the number of infiltrating immune cells, which were mainly composed of macrophages. Analysis of cytokines in the fluids of the pleural cavity lavages by suspension array indicated that levels of IL-2, IL-18, and IP-10 were significantly increased both at 24 h and at 3 months after the last spraying. In vitro proliferation assays revealed that a mixture of IL-2, IL-18, and IP-10, but not any of these cytokines alone, promoted cell proliferation of human fibroblasts and mesothelial cells. These results suggest that translocated and deposited MWCNTs induce subsequent pleural inflammation and that increased IL-2, IL-18, and IP-10 synergistically promote the development of pleural fibrosis and mesothelial proliferation.

This paper discusses the theoretical considerations for direction-of-arrival (DOA) estimation using antenna arrays in the presence of mutual coupling. In arrays, the relative proximity of antenna elements results in some manner of near-field mutual coupling that can negatively impact the array performance. In particular, mutual coupling can degrade the quality of DOA estimations and reduce the ability of the array to perform high-quality correlation processing and direction finding. The expected variance of an array performing DOA estimation is inversely related to the Fisher information matrix of the system. Theoretical radiated fields of a linear antenna array were compared to more realistic behavior of the equivalent architectures produced in electromagnetics simulation software. The mutual coupling between all the elements in an array can be a difficult phenomenon to precisely define, as it is an iterative process with many higher-order effects. To circumvent this, a mutual coupling matrix is defined as the relation between the theoretical radiation characteristic of an array and its simulated counterpart. An inverse solution method was used to solve for the mutual coupling interactions. The expected impact of mutual coupling in a DOA estimation context was then mathematically established by propagating the mutual coupling matrix through calculation of the Fisher information matrix and compared to the case of no mutual coupling. It was found that taking mutual coupling into consideration yields a higher Cramer-Rao Lower Bound and as a result a greater RMS angle error in a DOA estimation context. Mutual coupling was also found to have a somewhat greater impact on the Cramer-Rao Bound at S-band than at X-band.

Exposure to asbestos results in serious risk of developing lung and mesothelial diseases. Currently, there are no biomarkers that can be used to diagnose asbestos exposure. The purpose of the present study was to determine whether the levels or detection rate of chemokine (C-C motif) ligand 3 (CCL3) in the serum are elevated in persons exposed to asbestos. The primary study group consisted of 76 healthy subjects not exposed to asbestos and 172 healthy subjects possibly exposed to asbestos. The secondary study group consisted of 535 subjects possibly exposed to asbestos and diagnosed with pleural plaque (412), benign hydrothorax (10), asbestosis (86), lung cancer (17), and malignant mesothelioma (10). All study subjects who were possibly exposed to asbestos had a certificate of asbestos exposure issued by the Japanese Ministry of Health, Labour and Welfare. For the primary study group, levels of serum CCL3 did not differ between the two groups. However, the detection rate of CCL3 in the serum of healthy subjects possibly exposed to asbestos (30.2%) was significantly higher (P

Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.

Two types of nanosized titanium dioxide, anatase (anTiO2) and rutile (rnTiO2), are widely used in industry, commercial products and biosystems. TiO2 has been evaluated as a Group 2B carcinogen. Previous reports indicated that anTiO2 is less toxic than rnTiO2, however, under ultraviolet irradiation anTiO2 is more toxic than rnTiO2 in vitro because of differences in their crystal structures. In the present study, we compared the in vivo and in vitro toxic effects induced by anTiO2 and rnTiO2. Female SD rats were treated with 500 ?g/ml of anTiO2 or rnTiO2 suspensions by intra-pulmonary spraying 8 times over a two week period. In the lung, treatment with anTiO2 or rnTiO2 increased alveolar macrophage numbers and levels of 8-hydroxydeoxyguanosine (8-OHdG); these increases tended to be lower in the anTiO2 treated group compared to the rnTiO2 treated group. Expression of MIP1??mRNA and protein in lung tissues treated with anTiO2 and rnTiO2 was also significantly up-regulated, with MIP1??mRNA and protein expression significantly lower in the anTiO2 group than in the rnTiO2 group. In cell culture of primary alveolar macrophages (PAM) treated with anTiO2 and rnTiO2, expression of MIP1??mRNA in the PAM and protein in the culture media was significantly higher than in control cultures. Similarly to the in vivo results, MIP1??mRNA and protein expression was significantly lower in the anTiO2 treated cultures compared to the rnTiO2 treated cultures. Furthermore, conditioned cell culture media from PAM cultures treated with anTiO2 had less effect on A549 cell proliferation compared to conditioned media from cultures treated with rnTiO2. However, no significant difference was found in the toxicological effects on cell viability of ultra violet irradiated anTiO2 and rnTiO2. In conclusion, our results indicate that anTiO2 is less potent in induction of alveolar macrophage infiltration, 8-OHdG and MIP1??expression in the lung, and growth stimulation of A549 cells in vitro than rnTiO2.

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation–promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 μg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn2+. In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn2+ from the lung. Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1086-5) contains supplementary material, which is available to authorized users.

Summary The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP-dependent oncogenic H- or K-ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre-expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.

The performance of different random array geometries is analyzed and compared. Three phased array geometries are considered: linear arrays with non-uniform randomized spacing between elements, circular arrays with non-uniform element radii, and ad hoc sensor networks with elements located randomly within a circular area. For each of these array geometries, computer simulations modeled the transmission, reflection from an arbitrary target, and reception of signals. The effectiveness of each array’s beamforming techniques was measured by taking the peak cross-correlation between the received signal and a time-delayed replica of the original transmitted signal. For each array type, the correlation performance was obtained for transmission and reception of both chirp waveforms and ultra-wideband noise signals. It was found that the non-uniform linear array generally produced the highest correlation between transmitted and reflected signals. The non-uniform circular and ad hoc arrays demonstrated the most consistent performance with respect to noise signal bandwidth. The effect of scan angle was found to have a significant impact on the correlation performance of the linear arrays, where the correlation performance declines as the scan angle moves away from broadside to the array.

Lactoferrin is a major component of biologically important mucosal fluids and of the specific granules of neutrophils. Understanding its biological function is essential for understanding neutrophil- and mucosal-mediated immunity. In this review, we reevaluate the in vivo functions of human lactoferrin (hLF) emphasizing in vivo studies and in vitro studies performed in biologically relevant fluids. We discuss the evidence in the literature that supports (or does not support) proposed roles for hLF in mucosal immunity and in neutrophil function. We argue that the current literature supports a microbiostatic role, but not a microbicidal role, for hLF in vivo. The literature also supports a role for hLF in inhibiting colonization and infection of epithelial surfaces by microorganisms and in protecting tissues from neutrophil-mediated damage. Using this information, we briefly discuss hLF in the context of the complex biological fluids in which it is found.

Summary The membrane-anchored metalloproteinase-regulator RECK has been characterized as a tumor suppressor. Here we report that mice with reduced Reck-expression show limb abnormalities including right-dominant, forelimb-specific defects in postaxial skeletal elements. The forelimb buds of low-Reck mutants have an altered dorsal ectoderm with reduced Wnt7a and Igf2 expression, and hypotrophy in two signaling centers (i.e., ZPA and AER) that are essential for limb outgrowth and patterning. Reck is abundantly expressed in the anterior mesenchyme in normal limb buds; mesenchyme-specific Reck inactivation recapitulates the low-Reck phenotype; and some teratogens downregulate Reck in mesenchymal cells. Our findings illustrate a role for Reck in the mesenchymal-epithelial interactions essential for mammalian development.

Titanium dioxide (TiO(2)) is used in sunscreens and cosmetics as an ultraviolet light screen. TiO(2) has carcinogenic activity in the rat lung, but its effect on the skin has not been reported. We examined the promoting/carcinogenic effect of nano-size TiO(2) particles using a two-stage skin model. c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to ultraviolet B radiation on shaved back skin twice weekly for 10 weeks; then the shaved area was painted with a 100 mg/ml TiO(2) suspension twice weekly until sacrifice. All rats were killed at week 52 except for female Hras128 rats which were sacrificed at week 16 because of early mammary tumor development. Skin tumors developed in male Hras128 rats and mammary tumors developed in both sexes of Hras128 rats and in wild-type female rats, but tumor incidence was not different from controls. TiO(2) particles were detected in the upper stratum corneum but not in the underlying skin tissue layers. TiO(2) particles also did not penetrate a human epidermis model in vitro. Our data suggest that TiO(2) does not cause skin carcinogenesis, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.

Pancreatic ductal adenocarcinoma (PDA) is one of the most debilitating malignancies in humans. A thorough understanding of the cytogenesis of this disease will aid in establishing successful treatments. We have developed an animal model which uses adult Hras(G12V) and Kras(G12V) transgenic rats in which oncogene expression is regulated by the Cre/loxP system and neoplastic lesions are induced by injection of adenovirus-expressing Cre recombinase. When adenovirus with Cre recombinase under the control of the CMV enhancer/chicken beta-actin (CAG) promoter (Ad-CAG-Cre) is injected into the pancreatic duct of these animals, pancreatic neoplasias develop. Pathologically, the origin of these lesions is duct, intercalated duct, and centroacinar cells, but not acinar cells. The present study was undertaken to test the effect of acinar cell-specific oncogenic ras expression. Adult transgenic rats were injected with adenovirus with Cre recombinase under the control of the acinar cell-specific promoters amylase (Ad-Amy-Cre) and elastase-1 (Ad-Ela-Cre) or under the control of the non-specific CAG promoter. Injection of either Ad-Amy-Cre or Ad-Ela-Cre into the pancreatic ducts of transgenic animals in which oncogenic Kras is tagged with hemagglutinin (HA), HA-Kras(G12V) rats resulted in expression of oncogenic ras in acinar cells but not in duct, intercalated duct, or centroacinar cells. Notably, injected animals did not develop any observable proliferative or neoplastic lesions. In marked contrast, injection of Ad-CAG-Cre resulted in pancreatic cancer development within 4 weeks. These results indicate that adult acinar cells are refractory to Ras oncogene activation and do not develop neoplasia in this model.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. Animal PDA models which reflect the human condition are clearly necessary to develop early diagnostic tools and explore new therapeutic approaches. We have established transgenic rats carrying a mutated H- or K-ras gene (Hras250 and Kras327) controlled by Cre/loxP activation. These animals develop PDA which are histopathologically similar to that in humans. We utilized this model to identify biomarkers to detect early PDA. We report here that serum levels of Erc/Mesothelin are significantly higher in rats bearing PDA than in controls. Importantly, the levels are significantly elevated in rats before grossly visible carcinomas develop. Even in rats with very small microscopic ductal carcinoma lesions, elevated serum Erc/Mesothelin can be detected. We believe this is the first report of a pancreas tumor animal model in which pre-symptomatic lesions can be diagnosed.

Lactoferrin (LF), a secreted, iron binding glycoprotein originally discovered as a component of milk, is found in a variety of exocrine secretions and in the secondary granules of polymorphonuclear leukocytes. Animal experiments have shown that oral administration of bovine lactoferrin (bLF) exerts anticarcinogenesis effects in the colon and other organs of the rat. The aim of this study was to determine whether oral bLF could inhibit the growth of adenomatous colorectal polyps in human patients. A randomized, double-blind, controlled trial was conducted in 104 participants, ages 40 to 75 years, with polyps ≤5 mm in diameter and likely to be adenomas. Participants were assigned to receive placebo, 1.5-g bLF, or 3.0-g bLF daily for 12 months. Target adenomatous polyps were monitored by colonoscopy. Ingestion of 3.0-g bLF significantly retarded adenomatous polyp growth in participants 63 years old or younger. Removal of adenomatous colorectal polyps is done as a preventative measure against colorectal cancer; however, polyps can be overlooked, and when detected, polypectomy is not always 100% effective in eradicating a polyp. Our study suggests that daily intake of 3.0 g of bLF could be a clinically beneficial adjunct to colorectal polyp extraction.

Ingestion of arsenic (As) through contaminated drinking water results in increased risks of skin, lung, kidney, and bladder cancers. Due to its association with kidney and bladder cancers, we hypothesized that analysis of the urinary proteome could provide insight into the mechanisms of As toxicity. Urine from participants in a cross-sectional As biomarker study conducted in Nevada, classified as having either high (or= 100 microg total urinary As/l) or low exposure (100 microg total urinary As/l) was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Two polypeptides, 2.21 and 4.37 kDa, were significantly decreased in the high exposure group (p0.05) and were limited to men when stratified by sex. To replicate these findings, urine from participants in a second As study in Chile was analyzed and results confirmed the decrease of the 4.37 kDa polypeptide as well as a 4.76 kDa polypeptide among highly exposed men. These peaks were identified and confirmed as human beta-defensin-1 (HBD-1) peptides. In a separate in vitro experiment, gene expression analysis of As-treated cell lines demonstrated reduced HBD1 mRNA confirming that the observed decrease in HBD-1 resulted from As exposure. HBD-1 is an antimicrobial peptide constitutively expressed in multiple tissues including epithelial cells of the respiratory and urogenital systems. Recent studies support its role as a tumor suppressor gene for urological cancers suggesting that decreased HBD-1 levels may play a role in the development of cancers associated with As exposure. Further studies are warranted to investigate the role of HBD-1 in As-related toxicity.

This correspondence concerns a recent publication inCancer Cellby Liu et al.1 who analyzed a long noncoding RNA (lncRNA) that they designated “NKILA”. Liu et al. found thatNKILA(1) is upregulated by immunostimulants, (2) has a promoter with an NF-ĸB binding motif, (3) can bind to the p65 protein of the NF-ĸB transcription factor and then interfere with phosphorylation of IĸBα, and (4) negatively affects functions that involve NF-ĸB pathways. And, importantly, they found that (5) lowNKILAexpression in breast cancers is associated with poor patient prognosis. However, they entirely failed to mentionPMEPA1, a gene which runs antisense toNKILA, and the expression of which is associated with several tumors and which encodes a protein that participates in immune pathways.ThePMEPA1locus, including its promoter region, which Liu et al.1only discuss in regard toNKILA, is highly conserved through evolution. Our impression is thatNKILAemerged only later in evolution, possibly as an additional means ofPMEPA1regulation. Liu et al., however, only consider direct binding betweenNKILAand NF-ĸB as the mechanism for theirin vivoobservations ofNKILAfunction, but do not provide solid evidence for their model. Ifin vivoobservations by Liu et al. could be explained byNKILAregulation ofPMEPA1, it would contribute to the establishment ofPMEPA1as an important topic of cancer research. We feel that the herein presented discussion is necessary for a correct interpretation of the Liu et al. article.

Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of