MicroRNA Biomarkers: Pitfalls and Potential.

Thirty years ago, the small molecules we now know as microRNAs (miRNAs) would have been regarded as "junk" genetic material. Today, miRNAs have been hailed as promising biomarkers for diseases including cancer, neurological disorders, heart conditions, and infections. Although progress has been made toward characterizing the role of miRNAs in different diseases, there remain challenges in bringing them into clinical use. Researchers continue to explore where miRNAs will make the most impact.

The Cancer Moonshot Enters a New Phase.

The Obama Administration launched the Cancer Moonshot in 2016 to accelerate aspects of cancer research, with then-Vice President Biden in charge of "mission control." With the signing into law of the 21st Century Cures Act, the U.S. Congress authorized $ 1.8 billion in funding for the Cancer Moonshot over seven years starting in 2017. The National Cancer Institute (NCI) has invested those funds in more than 240 research projects and 70 programs in research priority areas including improving the understanding of cancer, encouraging collaboration and data sharing, and addressing cancer disparities [1].

The mRNA Revolution is Coming.

A bright spot during this COVID-19 pandemic has been the rapid development of effective vaccines that work by harnessing the power of messenger RNA, or mRNA. mRNA vaccines might seem like a relatively new idea, but researchers have been working on the technology behind them for decades. Now, the success of Moderna and Pfizer's coronavirus vaccines are highlighting the immense potential for mRNA therapies-not just for infectious diseases, but also to treat cancer and genetic disorders.

Stem Cell Update: Where Are We Now?

Since stem cells give rise to all the different cell types that make up our bodies, they have the potential to repair or replace cells that are missing or dysfunctional in a wide range of diseases and injuries. In recent years, an explosion of clinical trials involving stem cell therapies has inspired hope that such regenerative strategies may soon cure some of our most vexing diseases. Before that hope is realized, we will need a greater understanding of the fundamentals of stem cell biology as well as the specifics of different disease processes. Although the challenges seem daunting, stem cell research is rapidly advancing and ushering in a new era of regenerative medicine.

Direct-To-Consumer Genetic Testing: Is the Public Ready for Simple, At-Home DNA Tests to Detect Disease Risk?

Most genetic testing requires a doctor's prescription. In April 2017, however, the U.S. Food and Drug Administration (FDA) gave genetics company 23andMe the go-ahead to sell DNA tests assessing the user's level of risk for ten health conditions, including Parkinson's disease and late-onset Alzheimer's disease. This was followed nearly a year later by approval to sell tests for three mutations in the genes BRCA1 and BRCA2 linked to increased breast cancer risk. These remain the only FDA-approved direct-to-consumer (DTC) tests for genetic risk of disease.

Tackling an Epidemic: New and Emerging Opioid Addiction Treatments Offer Hope for Solutions to This Crisis.

The opioid epidemic is dominating news headlines and inspiring greater calls for political action in the United States. While opioid addiction isn't a new issue, its devastating consequences are now being felt across the country. "What's got everybody's attention is that so many people are dying," says Dr. Richard Ries (Figure 1), director of the University of Washington School of Medicine?s addictions divisions and medical director of the Outpatient Addictions Program at Harborview Medical Center, Seattle. "Currently, the top reason for early death in people under 50 is now overdose-more than cancer, AIDS, or accidents. The bulk of those overdoses involve opioids."

A New Fight Against Colon Cancer: As Cancer Rates Rise Among Younger Adults, Researchers Look for New Screening and Treatment Options.

Although the overall incidence of colon cancer has been falling over the past few decades, a pair of recent studies revealed a startling trend. In February 2017, researchers published a report showing that colon cancer rates were rising among younger adults. Some skeptics suggested the spike might simply reflect earlier detection and not necessarily represent a real increase in the disease. But a follow-up study found that Gen-Xers and millenials aren't just getting cancer diagnoses earlier; they are dying from colon cancer at slightly higher rates than in previous decades. Death rates among adults aged 20-54 have increased slightly, to 4.3 deaths per 100,000 people in 2014, up from 3.9 per 100,000 in 2004.

Technological Innovation Comes to Palliative Care: With a shortage of palliative specialists, telemedicine and remote monitoring offer relief.

At first, palliative care and technology might seem like strange bedfellows. At its core, palliative care is a very human side of medicine, relying heavily on talking with and listening to people to understand their experiences and goals. Technology, on the other hand, can often feel impersonal, cold, and one-size-fitsall. Despite this apparent disconnect, researchers and clinicians are finding new ways to harness technology to facilitate communication between patients and caregivers.

Human airway eosinophils respond to chemoattractants with greater eosinophil-derived neurotoxin release, adherence to fibronectin, and activation of the Ras-ERK pathway when compared with blood eosinophils.

Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras-ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras-ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras-ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.

Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.

The primary function of IL-7 is to promote maturation and survival of T cells. Through microarray expression analysis, we previously observed that human blood eosinophils express mRNA for IL-7R alpha (CD127) and its common gamma chain (CD132). The purpose of this study was to determine whether eosinophils have functional IL-7 receptors and to assess the potential contribution of IL-7 to eosinophilic airway inflammation by evaluating its presence in bronchoalveolar lavage (BAL) fluid of subjects with atopic asthma before and after segmental bronchoprovocation with allergen. Immunoblot analysis revealed that CD127 is present in highly purified human blood eosinophils. Furthermore, eosinophils responded to IL-7 with phosphorylation of STAT5, up-regulation of the activation marker CD69, and prolonged survival. Neutralization of GM-CSF but not IL-5 significantly blunted these functional responses, suggesting that IL-7 mediates its effects by promoting eosinophil release of autologous GM-CSF. Notably, the suppressive effect of anti-GM-CSF on STAT5 phosphorylation occurred within 10 min of eosinophil exposure to IL-7. Thus, IL-7 likely activates eosinophil release of preformed rather than newly synthesized GM-CSF. The biological relevance of IL-7 to eosinophilia in vivo was implicated in a study of airway allergen challenge in patients with allergic asthma. IL-7 concentrations in BAL fluid increased significantly 48 h after segmental allergen challenge and were highly correlated with BAL eosinophils (r = 0.7, p < 0.001). In conclusion, the airway response to allergen is associated with the generation of IL-7, which may contribute to airway inflammation by promoting enhanced eosinophil activation and survival. Activation of eosinophils is a novel function for IL-7.

Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-alpha.

Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-alpha plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma-inducible protein-10), were expressed after stimulation with TNF-alpha plus IFN-gamma. Addition of TNF-alpha appeared to be essential for IFN-gamma-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-kappaB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-kappaB, STAT6, and STAT1 was induced in eosinophils by TNF-alpha, IL-4, and IFN-gamma, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-alpha plus IL-4 or TNF-alpha plus IFN-gamma. Thus, independently activated signaling cascades appear to lead to activation of NF-kappaB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-alpha is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.

Flt3-L gene therapy enhances immunocytokine-mediated antitumor effects and induces long-term memory.

Therapeutic treatment with hu14.18-IL-2 immunocytokine (IC) or Flt3-L (FL) protein is initially effective at resolving established intradermal NXS2 neuroblastoma tumors in mice. However, many treated animals develop recurrent disease. We previously found that tumors recurring following natural killer (NK) mediated IC treatment show augmented MHC class I expression, while the tumors that recurred following T cell dependent Flt3-L treatment exhibited decreased MHC class I expression. We hypothesized that this divergent MHC modulation on recurrent tumors was due to therapy-specific immunoediting. We further postulated that combining IC and Flt3-L treatments might decrease the likelihood of recurrent disease by preventing MHC modulation as a mechanism for immune escape. We now report that combinatorial treatment of FL plus hu14.18-IL-2 IC provides greater antitumor benefit than treatment with either alone, suppressing development of recurrent disease. We administered FL by gene therapy using a clinically relevant approach: hydrodynamic limb vein (HLV) delivery of DNA for transgene expression by myofibers. Delivery of FL DNA by HLV injection in mice resulted in systemic expression of >10 ng/ml of FL in blood at day 3, and promoted up to a fourfold and tenfold increase in splenic NK and dendritic cells (DCs), respectively. Furthermore, the combination of FL gene therapy plus suboptimal IC treatment induced a greater expansion in the absolute number of splenic NK and DCs than achieved by individual component treatments. Mice that received combined FL gene therapy plus IC exhibited complete and durable resolution of established NXS2 tumors, and demonstrated protection from subsequent rechallenge with NXS2 tumor.

Hydrodynamic limb vein delivery of a xenogeneic DNA cancer vaccine effectively induces antitumor immunity.

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation.

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10), which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however, there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore, the involvement of resident airway immune cells, predominantly bronchoalveolar macrophages, in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard, our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release, which occurred in a time- and dose-dependent manner. Furthermore, HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo.

Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA.

Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by,transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.

The role of p38 MAPK in rhinovirus-induced monocyte chemoattractant protein-1 production by monocytic-lineage cells.

Viral respiratory infections are a major cause of asthma exacerbations and can contribute to the pathogenesis of asthma. Major group human rhinovirus enters cells by binding to the cell surface molecule ICAM-1 that is present on epithelial and monocytic lineage cells. The focus of the resulting viral infection is in bronchial epithelia. However, previous studies of the cytokine dysregulation that follows rhinovirus infection have implicated monocytic lineage cells in establishing the inflammatory environment even though productive infection is not a result. We have determined that human alveolar macrophages and human peripheral blood monocytes release MCP-1 upon exposure to human rhinovirus 16 (HRV16). Indeed, we have found p38 MAPK activation in human alveolar macrophages within 15 min of exposure to HRV16, and this activation lasts up to 1 h. The targets of p38 MAPK activation include transcriptional activators of the MCP-1 promoter. The transcription factor ATF-2, a p38 MAPK substrate, is phosphorylated 45 min after HRV16 exposure. Furthermore, IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, is degraded. Prevention of HRV16 binding was effective in blocking p38 MAPK activation, ATF-2 phosphorylation, and MCP-1 release. This is the first report of a relationship between HRV16 exposure, MCP-1 release and monocytic-lineage cells suggesting that MCP-1 plays a role in establishing the inflammatory microenvironment initiated in the human airway upon exposure to rhinovirus.

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils.

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.

Expression of interleukin-5- and granulocyte macrophage-colony-stimulating factor-responsive genes in blood and airway eosinophils.

Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor alpha subunit, lymphotoxin beta, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5- and GM-CSF-responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor alpha subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin beta and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge.

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process.

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.