Background Early and accurate diagnosis followed by timely treatment are the key prerequisites to fight tuberculosis (TB) and reduce its global burden. Despite scientific advances, the rapid and correct diagnosis of both pulmonary and extrapulmonary tuberculosis remains a challenge due to traditional reliance on detection of the elusive bacilli. Mycobacterium tuberculosis (Mtb)-specific host immune activation and cytokine production have shown significant promise as alternative means of detecting and distinguishing active disease from latent infection. We queried the diagnostic ability of phenotypic markers on Mtb-specific cytokine-producing immune cell subsets for identifying active tuberculosis. Methods Subjects belonging to the following groups were recruited – pulmonary and extrapulmonary TB, latent TB, cured TB, sick controls and healthy controls. Polychromatic flow cytometry was used to identify host immune biomarkers in an exploratory cohort comprising 56 subjects using peripheral blood mononuclear cells. Clinical performance of the identified biomarker was evaluated using whole blood in a blinded validation cohort comprising 165 individuals. Results Cytokine secreting frequencies of Mtb-specific CD4 + T cells with CD38 +CD27 – phenotype clearly distinguished infected individuals with active tuberculosis from those without disease. TNF-α secretion from CD38 +CD27 –CD4 + T cells upon stimulation with ESAT6/CFP10 peptides had the best diagnostic accuracy at a cut-off of 9.91% [exploratory: 96.67% specificity, 88.46% sensitivity; validation: 96.15% specificity, 90.16% sensitivity]. Additionally, this subset differentiated treatment-naive TB patients from individuals cured of TB following completion of anti-tuberculosis therapy. Conclusions Mtb-specific CD38 +CD27 –TNF-α +CD4 + T cell subset is a robust biomarker both for diagnosing tuberculosis and assessing cure.

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RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson crick (WC) base pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs) have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl) glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance-based mechanism. In the last three decades various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and non-coding RNAs for a myriad of therapeutic applications.

BackgroundEarly biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and “cytokine storm” associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue.ResultsDengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised.ConclusionOur findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ+CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85–0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.

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