Circulating microRNAs miR-331 and miR-195 differentiate local luminal a from metastatic breast cancer.

BACKGROUND: Breast cancer is the leading cause of cancer related death in women, with metastasis the principle cause of mortality. New non-invasive prognostic markers are needed for the early detection of metastasis, facilitating treatment decision optimisation. MicroRNA (miRNA) are small, non-coding RNAs regulating gene expression and involved in many cellular processes, including metastasis. As biomarkers, circulating miRNAs (in blood) hold great promise for informing diagnosis or monitoring treatment responses. METHODS: Plasma extracted RNA from age matched local Luminal A (n = 4) or metastatic disease (n = 4) were profiled using Next Generation Sequencing. Selected differentially expressed miRNA were validated on a whole blood extracted miRNA cohort [distant metastatic disease (n = 22), local disease (n = 31), healthy controls (n = 21)]. Area Under the Curve (AUC) in Receiver Operating Characteristic (ROC) analyses was performed. RESULTS: Of 4 miRNA targets tested (miR-181a, miR-329, miR-331, miR-195), mir-331 was significantly over-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p < 0.001). miR-195 was significantly under-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p = 0.043). In combination, miR-331 and miR-195 produced an AUC of 0.902, distinguishing metastatic from local breast cancer. CONCLUSIONS: We identified and validated two circulating miRNAs differentiating local Luminal A breast cancers from metastatic breast cancers. Further investigation will reveal the molecular role of these miRNAs in metastasis, and determine if they are subtype specific. This work demonstrates the ability of circulating miRNA to identify metastatic disease, and potentially inform diagnosis or treatment effectiveness.

Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379.

Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer.

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

Detailed analysis of a family of RNAs transcribed from varicella-zoster virus open reading frame 21 during replication.

The expression of VZV open reading frame (ORF) 21 during viral replication was examined at the RNA level. Three distinct mRNAs homologous to ORF 21, 3.6, 3.0, and 2.4 kb in size, were detected by Northern blot hybridization. Sequence analysis of eight ORF 21 cDNAs indicated that the corresponding RNAs were 3' coterminal and unspliced. The three largest cDNAs contained the entire ORF 21 coding sequence. Two upstream alternative polyadenylation signals located within ORF 21 were not utilized. Sequence analysis of the site of polyadenylation suggests a virus-specific cis-acting element is present in these transcripts.

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology.

The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5 X 10(7) cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.

Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia.

Epstein-Barr virus (EBV) DNA structure and gene expression were analyzed in tissue specimens from oral hairy leukoplakia (HLP), a mucocutaneous lesion that develops in patients infected with human immunodeficiency virus (HIV). The structure of the terminal restriction enzyme fragments of EBV revealed that HLP is a permissive infection without a predominant, detectable population of EBV episomal DNA. In RNA preparations from this uniquely permissive infection, EBV replicative mRNAs could be identified by Northern analysis; however, the virally encoded small nuclear RNAs, the EBERs, were not detected in most HLP RNA preparations. In situ hybridization detected EBER expression in very rare cells. These data indicate that unlike other viral small nuclear RNAs, the EBERs are not expressed during viral replication and must participate in the complex maintenance of latent EBV infection.

Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma.

Two species of the Epstein-Barr virus-encoded latent membrane protein 2, LMP2A and LMP2B, are generated by alternative splicing, each species having a distinct first exon. LMP2 transcription in undifferentiated nasopharyngeal carcinoma (NPC), which is consistently associated with the Epstein-Barr virus, was investigated. Fifteen NPC specimens were analyzed by Northern (RNA) blot and RNA-based polymerase chain reaction; the LMP2A transcript was present in all specimens except one. In some specimens the LMP2B transcript was also detected. Sequence analysis of LMP2 cDNAs obtained from two NPC specimens revealed four mutations in exon 1 of the LMP2A transcript, which were present in both tumors and which resulted in nonconservative amino acid changes. These data suggest that the LMP2 expressed in NPC is distinct from that which is expressed in lymphoid cells.

Identification of Staphylococcus aureus enterotoxins A and B genes by PCR-ELISA.

We developed PCR-enzyme linked immunosorbent (ELISA) assays to detect Staphylococcus aureus enterotoxins A and B genes. The assays use internal biotin-labelled oligonucleotides as capture probes for immobilizing and subsequently detecting target sequences on microtiter plates. The detection limits of the PCR-ELISAs were approximately 250 gene copies, versus 2500 gene copies by agarose gel analysis. The sensitivity of the assays, as determined from a reference panel of 46 coded samples that included DNA purified from 31 different bacterial species and strains, SEA and SEB plasmid controls, and no-template controls was 100%. No cross-reactivity was observed with DNA from non-staphylococcal species. Using 27 clinical isolates of S. aureus, the SEA PCR-ELISA identified the enterotoxin A (sea) gene in 26 samples, and the SEB PCR-ELISA identified the enterotoxin B (seb) gene in all 27 samples. Compared with conventional antigen capture ELISAs for SEA and SEB toxins, the PCR-ELISAs showed overall superior detection limits. The sensitivity and specificity levels of the SEA PCR-ELISA and the SEA toxin ELISA were comparable within their respective detection thresholds, but the sensitivity and specificity of the SEB PCR-ELISA was much greater than that of SEB toxin ELISA.

Novel transcription from the Epstein-Barr virus terminal EcoRI fragment, DIJhet, in a nasopharyngeal carcinoma.

Transcription of Epstein-Barr virus (EBV) genes in epithelial tissue, one of the two principal cell types infected by EBV, is not well characterized. EBV transcription in a nasopharyngeal carcinoma established in nude mice, C15, has been analyzed by using strand-specific RNA probes and sequence analysis of a C15 cDNA library. In C15, two equally abundant mRNAs of 3.7 and 2.8 kilobases (kb) are encoded by the sequences that encode latent membrane protein (LMP). Hybridization with probes specific for the 3' end of the LMP mRNA to Northern (RNA) blots and sequence analysis of cDNAs representing the messages indicated that the 3.7- and 2.8-kb mRNAs are 3' coterminal. Sequence analysis of additional cDNAs revealed an mRNA that is spliced identically to the LMP mRNA but is initiated 5' to the promoter for LMP. A probe representing the sequences contained within the cDNA which are 5' to the LMP promoter identified the 3.7-kb mRNA in C15 and a low-abundance 3.7-kb mRNA in B95-8 RNA. These data indicate that transcription of the LMP-encoding sequences is complex and that LMP can be expressed from an additional RNA in both nasopharyngeal carcinoma and lymphoid cells. Hybridization with BamHI-A identified a predominant 4.8-kb mRNA and two less abundant larger-molecular-weight mRNAs transcribed in C15. These mRNAs are consistently expressed in all passages in nude mice of the C15 tumor. Hybridization with strand-specific probes and sequence analysis of three cDNAs revealed that these mRNAs are transcribed from left to right. Sequence analysis of cDNAs representing the 3' end of the mRNAs identified an open reading frame that could potentially encode a protein of 174 amino acids. In situ hybridization of a 35S-labeled RNA probe homologous to the BamHI-A cDNA to tissue sections revealed that the BamHI-A mRNA is not focally expressed and is transcribed in all cells within the C15 tumor. Linear forms of EBV DNA were not detected in any of the C15 tumors, and replicative viral antigens have not been detected. These data suggest that the C15 tumor represents a latently infected tumor and that the transcription from BamHI-A, which is expressed in all cells, is not associated with virus replication.

Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo.

A family of mRNAs that are transcribed rightward through the BamHI A fragment have been detected in C15, a nasopharyngeal carcinoma (NPC) which has been passaged in nude mice. Northern (RNA) blot hybridizations indicate that these RNAs are also expressed in three other NPCs which have been established in nude mice and in an NPC obtained at biopsy. Moreover, hybridization in situ detected transcription from BamHI A in 12 NPCs and 1 Epstein-Barr virus (EBV)-containing carcinoma of the parotid gland. In each case, transcription was detected in all of the malignant epithelial cells. Transcription was not detected in two cases of EBV-positive lymphoma biopsies by in situ hybridization nor in latently infected EBV-positive lymphoblastoid cell lines by Northern blot hybridization. The consistent transcription of these sequences in latently infected epithelial malignancy but not in lymphoid cells suggests that this viral function is associated with latent EBV infection of epithelial cells. Sequence analysis of a cDNA synthesized from the C15 tumor, representing the 3' end of BamHI A messenger RNA, revealed an open reading frame (ORF). Translation of this ORF in vitro produced several peptides that were immunoprecipitated with antisera from patients with NPC. The detection of antibodies to the protein encoded by the ORF present in the BamHI A cDNA indicates that BamHI A encodes a protein which is expressed in vivo and is antigenic.

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins.

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP(Sc)) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrP(Sc) by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method. RESULTS: PrP(Sc) partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrP(Sc) and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP(Sc) and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP(Sc) and TSE infectivity. PrP(Sc) partitioning coincided with infectivity partitioning, which showed a close relationship between PrP(Sc) and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP(Sc) was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.