Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and ...therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.
Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of ...miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.
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1B3 is a mimic of the naturally occurring miRNA-193a-3p, a gene regulator that has potent tumor suppressor functions. We characterized the effect of 1B3 on gene expression in six human cancer cell lines. 1B3 was shown to strongly activate tumor suppressor pathways while inhibiting oncogenic cell signaling.
Despite the widespread use of bacillus Calmette-Guérin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated ...and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Guérin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette-Guérin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Guérin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.
Tests based on tuberculin purified protein derivative (PPD) cannot distinguish between tuberculosis infection, Mycobacterium bovis BCG vaccination, or exposure to environmental mycobacteria. The ...present study investigated the diagnostic potential of two Mycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle. Both antigens were frequently recognized in vivo and in vitro based on the induction of delayed-type hypersensitivity responses and the ability to induce gamma interferon production by lymphocytes, respectively. The combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) than PPD (7%).
As cancer is a multifactorial disease, the multimodal action of microRNAs makes them an attractive tool for novel therapeutic approaches. The tumor suppressive miR-7-5p has been shown to act on many ...aspects of oncogenesis, including cell proliferation, migration and angiogenesis, by targeting a spectrum of key genes. We developed a synthetic chemically modified miR-7-5p mimic, 5A2, and performed a comprehensive functional characterization in a panel of human cancer cell lines. 5A2 reduced cell proliferation in most cell lines by inducing cell cycle arrest. To enable systemic delivery of 5A2 to tumors, it was formulated in a novel lipid nanoparticle (INT-5A2) and we demonstrated the anti-tumor activity of INT-5A2 in an experimental human liver tumor-bearing mouse model. Next, RNA-sequencing was used to gain more insight into the molecular mechanism of action of 5A2 and demonstrated a broad repression of target mRNAs. Interestingly, Ingenuity Pathway Analysis revealed a new role for 5A2 in metabolic pathways. Validation experiments
in vitro
showed that 5A2 reduced the expression of key glycolysis and glutaminolysis enzymes, leading to a decrease in glycolysis, lactate secretion and intracellular glutamate availability. Taken together, these data strongly suggest that miR-7-5p/5A2 is a potent tumor suppressor that targets various key cellular pathways across cancer types. Therefore, 5A2 may represent a promising novel treatment strategy in oncology.
It is estimated that one‐third of the world's population is infected with Mycobacterium tuberculosis, but that only 10% of infected people break down with the disease. In the remaining 90% the ...infection remains clinically latent. In the present study, the immune mechanisms controlling the latent phase of tuberculosis infection were evaluated in a mouse model of latency and reactivation. Mice aerosol‐infected with M. tuberculosis were treated with anti‐mycobacterial drugs resulting in very low, stable bacterial numbers (<500 CFU in the spleen and lung) for 10–12 weeks followed by reactivation of the disease with increasing bacterial numbers. During latency, pathological changes in the lung had almost completely resolved and lymphocyte number and turnover were at the pre‐infection level. The CD4 subset was highly active during the acute phase of infection and could be detected by intracellular staining for IFN‐γ as well as after antigen‐specific stimulation with mycobacterial antigens. The CD8 subset was not involved in the acute stage of infection, but this subset was active and produced IFN‐γ during the latent phase of infection. In vivo depletion of T cell subsets supported these findings with a 6–7‐fold increase in bacterial numbers in the lung following anti‐CD4 treatment during the acute phase, while anti‐CD8 treatment did not have an effect. The opposite was found during the latent phase where anti‐CD8 treatment as well as anti‐IFN‐γ treatment both resulted in a 10‐fold increase in bacterial numbers in the lung, while anti‐CD4 treatment induced only a modest change.
The local and systemic immune response to rotavirus was investigated, both in previously exposed adult sheep and gnotobiotic lambs. Vaccines composed of rotavirus or recombinant VP6 mixed with or ...incorporated into ISCOMs were used to examine if one oral dose could induce a mucosal immune response and protection against subsequent challenge. Gnotobiotic lambs were vaccinated orally either with PBS/ISC, inactivated rotavirus (IRV)/ISC, recombinant VP6/ISC, or IRV alone, challenged 3 weeks later with a live virulent lamb strain, and killed 8-9 days after challenge. The immune response was measured as described above. The rotavirus-vaccinated groups had RV IgG ASC and antibodies in blood from 7 and 11 days respectively. After challenge, rotavirus-vaccinated groups cleared the virus in a reduced period (6-7 days vs 8-9 days), however this was only significant (p<0.05) in the IRV/ISC group. RV IgA antibodies were observed in serum and nasal secretions from 4 days after challenge. In intestinal scrapings, these were significantly (p<0.05) higher in the IRV/ISC and IRV groups. RV IgG antibodies were significantly (p<0.05) increased in nasal secretions and intestinal scrapings in the IRV/ISC and IRV groups. Lymphocytes proliferated significantly in JPPs against rotavirus in the IRV/ISC and IRV groups. CD45R+ cells were significantly increased in blood in the IRV/ISC group before challenge, however no differences were found in other lymphocyte subpopulations either in blood or GALT. A downregulation of IFNγ transcripts was observed in JPPs and MLNs in the IRV/ISC group while the VP6/ISC group had a higher expression compared with the PBS/ISC and IRV groups. IL-4 transcripts were low in MLNs in all groups but in JPPs the rotavirus-vaccinated groups had a higher expression. IL-6 transcripts in JPPs were higher in the IRV/ISC and VP6/ISC groups but in MLNs all rotavirus-vaccinated groups had a higher level of expression. One oral dose of inactivated rotavirus alone, mixed with ISCOMs, or recombinant VP6 incorporated into ISCOMs, can induce priming and partial protection. These results suggest also that different immunological mechanisms take place when different vaccination protocols are used.
The local and systemic immune response to rotavirus was investigated, both in previously exposed adult sheep and gnotobiotic lambs. Vaccines composed of rotavirus or recombinant VP6 mixed with or ...incorporated into ISCOMs were used to examine if one oral dose could induce a mucosal immune response and protection against subsequent challenge. Gnotobiotic lambs were vaccinated orally either with PBS/ISC, inactivated rotavirus (IRV)/ISC, recombinant VP6/ISC, or IRV alone, challenged 3 weeks later with a live virulent lamb strain, and killed 8-9 days after challenge. The immune response was measured as described above. The rotavirus-vaccinated groups had RV IgG ASC and antibodies in blood from 7 and 11 days respectively. After challenge, rotavirus-vaccinated groups cleared the virus in a reduced period (6-7 days vs 8-9 days), however this was only significant (p<0.05) in the IRV/ISC group. RV IgA antibodies were observed in serum and nasal secretions from 4 days after challenge. In intestinal scrapings, these were significantly (p<0.05) higher in the IRV/ISC and IRV groups. RV IgG antibodies were significantly (p<0.05) increased in nasal secretions and intestinal scrapings in the IRV/ISC and IRV groups. Lymphocytes proliferated significantly in JPPs against rotavirus in the IRV/ISC and IRV groups. CD45R+ cells were significantly increased in blood in the IRV/ISC group before challenge, however no differences were found in other lymphocyte subpopulations either in blood or GALT. A downregulation of IFNγ transcripts was observed in JPPs and MLNs in the IRV/ISC group while the VP6/ISC group had a higher expression compared with the PBS/ISC and IRV groups. IL-4 transcripts were low in MLNs in all groups but in JPPs the rotavirus-vaccinated groups had a higher expression. IL-6 transcripts in JPPs were higher in the IRV/ISC and VP6/ISC groups but in MLNs all rotavirus-vaccinated groups had a higher level of expression. One oral dose of inactivated rotavirus alone, mixed with ISCOMs, or recombinant VP6 incorporated into ISCOMs, can induce priming and partial protection. These results suggest also that different immunological mechanisms take place when different vaccination protocols are used.
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