40 Years of Pfs48/45 Research as a Transmission-Blocking Vaccine Target of Plasmodium falciparum Malaria.

In the early 1980s, Richard Carter was among the first researchers to identify the sexual stage-specific Pfs48/45 protein, leading to the identification of target epitopes. Carter predicted its tertiary conformation while involved in a number of studies on naturally acquired sexual stage-specific antibodies. Pfs48/45 is a cysteine-rich surface protein of sexual stages of Plasmodium falciparum that plays a critical role in male gamete fertility. Antibodies against Pfs48/45 prevent parasite development in the mosquito vector, and therefore prevent the spread of malaria in the population. Since the gene was sequenced in the early 1990s, Pfs48/45 has been considered a prime target candidate for a malaria transmission-blocking vaccine. However, major manufacturing challenges-in particular, difficulty realizing satisfactory yields of a properly folded protein for the induction of functional antibodies-delayed clinical development significantly. These challenges were met roughly 20 years later. The first clinical trial with a Pfs48/45 subunit vaccine (R0.6C) was started in the Netherlands in early 2021. The excellent contributions to the long and winding path of Pfs48/45 research by Richard Carter are well recognized and are an integrated part of his seminal contributions to unraveling Plasmodium sexual stage biology.

A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system.

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. However, due to its size and complexity, expression of the full-length protein has been difficult, leading to focus on the C-terminal six cysteine domain (6C) with the use of fusion proteins to facilitate expression and folding. In this study, we utilized the baculovirus system to evaluate the expression of three Pfs48/45 proteins including the full-length protein, the 6C domain fragment and the 6C domain mutant to prevent glycosylation. Expression of the recombinant Pfs48/45 proteins was conducted in super Sf9 cells combined with the use of tunicamycin to prevent N-glycosylation. The proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (SMFA). Only the 6C protein was found to exhibit significant transmission-reducing activity. Further characterization of the biologically active 6C protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. This study presents an alternative expression system, without the need of a fusion protein partner, for the Pfs48/45 6C protein fragment including further evaluation as a potential transmission-blocking vaccine candidate.

A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine.

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.

Peptide data on the disulfide bond analysis of baculovirus produced Pfs25 by LC-MSMS.

This article contains the peptide data obtained while performing disulfide bond mapping of the recombinant Plasmodium falciparum protein, Pfs25, produced from the baculovirus expression system. Pfs25 is a malaria transmission-blocking vaccine candidate, with a compact and complex structure including 22 cysteines. This supplementary data is related to the research "Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate" (Lee et al., 2018) [1]. In brief, Pfs25 was digested with trypsin/Lys-C and derived peptides separated by High Performance Liquid Chromatography (HPLC) and analyzed by mass spectrometry (MS) by MSE fragmentation. The theoretical peptides and their respective masses along with disulfide bond locations with linked peptides are presented here alongside the mass spectrometry analysis. The raw mass spectrometry data is made available through the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.

Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue.

The demand for nucleic acid and protein derivatives from formalin-fixed paraffin-embedded (FFPE) tissue has greatly increased due to advances in extraction and purification methods, making these derivatives available for numerous genomic and proteomic platforms. Previously, DNA, RNA, microRNA (miRNA), or protein derived from FFPE tissue blocks were considered "unfit" for such platforms, as the process of tissue immobilization by FFPE resulted in cross-linked, fragmented, and chemically modified macromolecules. We conducted a systematic examination of nucleic acids and proteins co-extracted from 118 FFPE blocks sampled from the AIDS and Cancer Specimen Resource (ACSR) at The George Washington University after stratification by storage duration and the three most common tumor tissue types at the ACSR (adenocarcinoma, squamous cell carcinoma, and papillary carcinoma). DNA, RNA, miRNA, and protein could be co-extracted from 98% of the FFPE blocks sampled, with DNA and miRNA "fit" for diverse genomic purposes including sequencing. While RNA was the most labile of the FFPE derivatives, especially when assessed by RNA integrity number (RIN), it was still "fit" for genomic methods that use smaller sequence lengths, e.g., quantitative PCR. While more than half of the protein derivatives were fit for proteomic purposes, our analyses indicated a significant interaction effect on the absorbance values for proteins derived from FFPE, implying that storage duration may affect protein derivatives differently by tumor tissue type. The mean absorbance value for proteins derived from more recently stored FFPE was greater than protein derived from older FFPE, with the exception of adenocarcinoma tissue. Finally, the fitness of one type of derivative was weakly associated with the fitness of derivatives co-extracted from the same FFPE block. The current study used several novel quality assurance approaches and metrics to show that archival FFPE tissue blocks are a valuable resource for contemporary genomic and proteomic platforms.

N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

Transmission-blocking vaccines have the potential to accelerate malaria parasite elimination by inducing antibodies that block parasite transmission from humans to mosquitoes. Pfs230, a gametocyte surface protein involved in gamete function, has long been a promising candidate. Due to the large size (3,135 amino acids), complex domains, and repeating 6-cysteine (6-Cys) motifs with a multitude of disulfide bonds, the feasibility of expression of a full-length protein has been difficult. A priority focus, therefore, has been on the generation of single domains, including N-terminal fragments. Here we utilized a heterologous expression system, baculovirus, to produce an N-terminal domain of Pfs230 (Pfs230C1). Pfs230C1 (amino acids 443 to 731) with a polyhistidine affinity tag was expressed in Super Sf9 cells. Since the native host lacks glycosylation machinery, a single N585Q mutation was made to eliminate potential N-linked glycosylation. The expressed protein, purified by nickel affinity, ion exchange, and size exclusion chromatography to >90% purity, was present in monomeric form with an observed mass of 33,510 Da (matching oxidized form). Peptide mapping and disulfide analysis confirmed the proper formation of predicted disulfide bonds. Antibodies, generated against Pfs230C1 in mice, bound to the gametocyte in an immunofluorescence assay (IFA) and demonstrated functional activity in both the standard membrane feeding assay (SMFA) and the exflagellation assay (EXA). The biochemical, biophysical, and immunological results reported herein support the continued advancement of an N-terminal Pfs230 antigen (Pfs230C1) as a component of a transmission-blocking vaccine. Our results also support the continued use of the scalable baculovirus expression system for the generation of complex Plasmodium proteins.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission-blocking vaccine candidates.

BACKGROUND: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery. METHODS: While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay. RESULTS: Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production. CONCLUSIONS: In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.

New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74.

Na-APR-1(M74) is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1(M74) and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1 wt in vitro, thereby providing proof of concept of Na-APR-1(M74) as a vaccine antigen. The mutated version, Na-APR-1(M74), was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1(M74) IgG. As little as 0.99 µg of recombinant Na-APR-1(M74) could induce anti Na-APR-1(M74) IgG in mice that were capable of inhibiting Na-APR-1w t-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1(M74) as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1 wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1(M74) which was assessed by fluorescence polarization, but with an ∼ 50-fold reduction in the dissociation constant. Taken together, these assays comprise a "toolbox" that could be useful for the analyses of Na-APR-1(M74) as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline.

A microRNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive tumor of the bile duct, and a significant public health problem in East Asia, where it is associated with infection by the parasite Opisthorchis viverrini. ICC is often detected at an advanced stage and with a poor prognosis, making a biomarker for early detection a priority. METHODS: We have comprehensively profiled miRNA expression levels in ICC tumor tissue using small RNA-Seq and validated these profiles using quantitative PCR on matched plasma samples. RESULTS: Distinct miRNA profiles were associated with increasing histological differentiation of ICC tumor tissue. We also observed that histologically normal tissue adjacent to ICC tumor displayed miRNA expression profiles more similar to tumor than liver tissue from healthy donors. In plasma samples, an eight-miRNA signature associated with ICC, regardless of the degree of histological differentiation of its matched tissue, forming the basis of a circulating miRNA-based biomarker for ICC. CONCLUSIONS: The association of unique miRNA profiles with different ICC subtypes suggests the involvement of specific miRNAs during ICC tumor progression. In plasma, an eight-miRNA signature associated with ICC could form the foundation of an accessible (plasma-based) miRNA-based biomarker for the early detection of ICC.

Circumventing qPCR inhibition to amplify miRNAs in plasma.

BACKGROUND: Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance. FINDINGS: Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures. CONCLUSION: Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.

Distinct miRNA signatures associate with subtypes of cholangiocarcinoma from infection with the tumourigenic liver fluke Opisthorchis viverrini.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a significant public health problem in East Asia, where it is strongly associated with chronic infection by the food-borne parasite Opisthorchis viverrini (OV). We report the first comprehensive miRNA expression profiling by microarray of the most common histologic grades and subtypes of ICC: well differentiated, moderately differentiated, and papillary ICC. METHODS: MicroRNA expression profiles from FFPE were compared among the following: ICC tumour tissue (n = 16), non-tumour tissue distally macrodissected from the same ICC tumour block (n = 15), and normal tissue (n = 13) from individuals undergoing gastric bypass surgery. A panel of deregulated miRNAs was validated by qPCR. RESULTS: Each histologic grade and subtype of ICC displayed a distinct miRNA profile, with no cohort of miRNAs emerging as commonly deregulated. Moderately differentiated ICC showed the greatest miRNA deregulation in quantity and magnitude, followed by the papillary subtype, and then well differentiated ICC. Moreover, when ICC tumour tissues were compared to adjacent non-tumour tissue, similar miRNA dysregulation profiles were observed. CONCLUSIONS: We show that common histologic grades and subtypes of ICC have distinct miRNA profiles. As histological grade and subtypes are associated with ICC aggressiveness, these profiles could be used to enhance the early detection and improve the personalised treatment for ICC. These findings also suggest the involvement of specific miRNAs during ICC tumour progression and differentiation. We plan to use these insights to (a) detect these profiles in circulation and (b) conduct functional analyses to decipher the roles of miRNAs in ICC tumour differentiation.

Methods and matrices: approaches to identifying miRNAs for nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC. METHODS: We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set. RESULTS: Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating "unknown" miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC. CONCLUSIONS: We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.

The miRNAome of Opisthorchis viverrini induced intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer, arising in the biliary ducts that extend into the liver. The highest incidence of ICC occurs in Southeast Asia, particularly in the Mekong River Basin countries of Thailand, Laos, Cambodia, and Vietnam, where it is strongly associated with chronic infection by the food-borne liver fluke Opisthorchis viverrini (OV), one of only three eukaryote pathogens considered Group one carcinogens. Intrahepatic cholangiocarcinoma is usually diagnosed at an advanced stage, with a poor prognosis and survival often less than 24 months. Hence, biomarkers that enable the early detection of ICC would be desirable and have a potentially important impact on the public health in the resource-poor regions where this cancer is most prevalent. As microRNAs (miRNAs) remain well preserved after formalin fixation, there is much interest in developing them as biomarkers that can be investigated using tumor biopsy samples preserved in formalin fixed paraffin embedded (FFPE) tumor blocks. Recently, we reported the first comprehensive profiling of tissue-based miRNA expression using FFPE from the three most common subtypes of OV-induced ICC tumors: moderately differentiated ICC, papillary ICC, and well-differentiated ICC. We observed that each subtype of OV-induced ICC exhibited a distinct miRNA profile, which suggested the involvement of specific sets of miRNAs in the progression of this cancer. In addition, non-tumor tissue adjacent to ICC tumor tissue on the same FFPE block shared a similar miRNA dysregulation profile with the tumor tissue than with normal (non-tumor) liver tissue (individuals without ICC or OV infection). Herein, we provide a detailed description of the microarray analysis procedures used to derive these findings.

Profiling miRNAs in nasopharyngeal carcinoma FFPE tissue by microarray and Next Generation Sequencing.

Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous-cell carcinoma that occurs in the epithelial lining of the nasopharynx. Nasopharyngeal carcinoma has a geographically well-defined distribution worldwide, with the highest prevalence in China, Southeast Asia, and Northern Africa. Symptoms of nascent NPC may be unapparent or trivial, with diagnosis based on the histopathology of biopsied tissue following endoscopy of the nasopharynx. The tumor node metastasis (TNM) staging system is the benchmark for the prognosis of NPC and guides treatment strategy. However, there is a consensus that the TNM system is not sufficiently specific for the prognosis of NPC, as it does not reflect the biological heterogeneity of this tumor, making another biomarker for the detection of NPC a priority. We have previously reported on different approaches for microRNA (miRNA) biomarker discovery for Formalin Fixed Paraffin Embedded (FFPE) NPC tissue samples by both a targeted (microarray) and an untargeted (small RNA-Seq) discovery platform. Both miRNA discovery platforms produced similar results, narrowing the miRNA signature to 1-5% of the known mature human miRNAs, with untargeted (small RNA-Seq approach) having the advantage of indicating "unknown" miRNAs associated with NPC. Both miRNA profiles strongly associated with NPC, providing two potential discovery platforms for biomarker signatures for NPC. Herein, we provide a detailed description of the methods that we used to interrogate FFPE samples to discover biomarkers for NPC.

Infection with the carcinogenic liver fluke Opisthorchis viverrini modifies intestinal and biliary microbiome.

Opisthorchis viverrini is a fish-borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tre where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke-induced CCA. Microbial communities were characterized by high-throughput sequencing of variable regions 7-9 of prokaryotic 16S ribosomal DNA. Of ∼1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to 20 phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P≤0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke-associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated IL-6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.

An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection.

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR-1. Ac-APR-1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood-feeding. Toward developing a human hookworm vaccine, we expressed both wild-type (Na-APR-1(wt)) and mutant (Na-APR-1(mut)-mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1(wt) was catalytically active, and Na-APR-1(mut) was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1(mut) and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na-APR-1(mut) induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1(wt) and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1(mut) was the most prominently detected antibody in sera from people resident in high-transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na-APR-1(mut) is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.

Thymosin fraction-5 possesses antiproliferative properties in HL-60 human promyelocytic leukemia cells: characterization of an active peptide.

Thymosin fraction-5 (TF5) is a protein preparation of the bovine thymus. TF5 stimulates many assays of T cell-mediated immunity. We found that TF5 substantially suppressed proliferation of the rat C6 glioma and MMQ pituitary adenoma cell lines. Our current research using the promyelocytic cell line HL-60 suggests that TF5 also prevents proliferation of human myeloid leukemia cells. Our objective is the purification and chemical characterization of TF5 peptide components responsible for inhibition of HL-60 proliferative capacity. Using the inhibition of HL-60 cell proliferation, we have chemically characterized TF5 using fast protein liquid chromatography (FPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and high-performance capillary electrophoresis (HPCE). Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were used to determine rates of HL-60 proliferation. Our results identified an approximately 6000 Da component of TF5 capable of inducing HL-60 growth arrest. Synchronized HL-60 cells exposed to TF5 and its various constituents were subjected to cytometric analysis by flow cytometry. TF5-treated HL-60 cells had an increased subdiploid faction (i.e., sub-G1) compared to control cells. TF5 also increased Annexin V staining in randomly cycling HL-60 cells. Thus, a TF5 subfraction possesses growth-suppressive activity for human myeloid neoplasms. Our results indicate that this effect is characterized by at least one hallmark of apoptosis. Future clinical management strategies for certain leukemias may involve the use of thymic peptides.