Neutrophils are the first infiltrating cell type essential for combating pneumoseptic infections by bacterial pathogens including Klebsiella pneumoniae (KPn). Following an infection or injury, removal of apoptotic infiltrates via a highly regulated process called efferocytosis is required for restoration of homeostasis, but little is known regarding the effect of bacterial infection on this process. Here we demonstrate that KPn infection impedes the efferocytic uptake of neutrophils in-vitro and in-vivo in lungs by macrophages. This impaired efferocytosis of infected neutrophils coincides with drastic reduction in the neutrophil surface exposure of apoptosis signature phospholipid phosphatidyserine (PS); and increased activity of phospholipid transporter flippases, which maintain PS in the inner leaflet of plasma membrane. Concomitantly, pharmacological inhibition of flippase activity enhanced PS externalization and restored the efferocytosis of KPn infected neutrophils. We further show that KPn infection interferes with apoptosis activation and instead activates non-apoptotic programmed cell death via activation of necroptosis machinery in neutrophils. Accordingly, pharmacological inhibition of necroptosis by RIPK1 and RIPK3 inhibitors restored the efferocytic uptake of KPn infected neutrophils in-vitro. Importantly, treatment of KPn infected mice with necroptosis inhibitor improved the disease outcome in-vivo in preclinical mouse model of KPn pneumonia. To our knowledge, this is the first report of neutrophil efferocytosis impairment by KPn via modulation of cell death pathway, which may provide novel targets for therapeutic intervention of this infection. Inflammatory diseases caused by infectious or sterile injuries are often characterized by pathological accumulation of dead or dying infiltrating cells. Pneumonic sepsis caused by Klebsiella pneumoniae (KPn), an opportunistic pathogen, has similar etiology, however, the underlying mechanism remains unknown. Here we report that KPn infection subverts a protective host process termed efferocytosis, by which the phagocytic cells engulf and clear dead/dying cells thereby resolving inflammation and infection. Our results show that KPn infected neutrophils are cleared less efficiently via efferocytosis than the uninfected neutrophils. Mechanistic studies implicated a reduced exposure of “eat me” signal phosphatidyleserine (PS) via increased flippase activity and skewing of cell death pathway toward necroptosis in impaired efferocytosis of infected neutrophils. Accordingly, pharmacological reversal of PS exposure by flippase inhibition, treatment with necroptosis inhibitors restored the efferocytic clearance of KPn infected neutrophils, and improved the disease outcome in a preclinical model of pneumonic sepsis. To our knowledge this is the first report of KPn subversion of efferocytic clearance of neutrophils by impairing pro-efferocytic apoptotic signatures and activation of necroptosis machinery. This could lead to novel therapeutic targets against KPn infection and associated inflammation in pneumonic sepsis.

The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has severely impacted human health and the health management system globally. The ongoing pandemic has required the development of more effective diagnostic strategies for restricting deadly disease. For appropriate disease management, accurate and rapid screening and isolation of the affected population is an efficient means of containment and the decimation of the disease. Therefore, considerable efforts are being directed toward the development of rapid and robust diagnostic techniques for respiratory infections, including SARS-CoV-2. In this article, we have summarized the origin, transmission, and various diagnostic techniques utilized for the detection of the SARS-CoV-2 virus. These higher-end techniques can also detect the virus copy number in asymptomatic samples. Furthermore, emerging rapid, cost-effective, and point-of-care diagnostic devices capable of large-scale population screening for COVID-19 are discussed. Finally, some breakthrough developments based on spectroscopic diagnosis that could revolutionize the field of rapid diagnosis are discussed.

Apolipophorin III (ApoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune responses of insects. Here we report the molecular and functional characterization of Anopheles stephensi Apolipophorin-III (AsApoLp-III) gene. This gene consists of 679 nucleotides arranged into two exons of 45 bp and 540 bp that give an ORF encoding 194 amino acid residues. Excluding a putative signal peptide of the first 19 amino acid residues, the 175-residues in mature AsApoLp-III protein has a calculated molecular mass of 22 kDa. Phylogenetic analysis revealed the divergence of mosquitoes (Order Diptera) ApoLp-III from their counterparts in moths (Order: Lepidoptera). Also, it revealed a close relatedness of AsApoLp-III to ApoLp-III of An. gambiae. AsApoLp-III mRNA expression is strongly induced in Plasmodium berghei infected mosquito midguts suggesting its crucial role in parasite development. AsApoLp-III silencing decreased P. berghei oocysts numbers by 7.7 fold against controls. These effects might be due to the interruption of AsApoLp-III mediated lipid delivery to the developing oocysts. In addition, nitric oxide synthase (NOS), an antiplasmodial gene, is also highly induced in AsApoLp-III silenced midguts suggesting that this gene acts like an agonist and protects Plasmodium against the mosquito immunity.

Purpose: Immune checkpoint therapy (ICT) is currently ineffective in a majority of patients. Tumor drug exposure measurements can provide vital insights into mechanisms involved in the resistance of solid tumors to those therapeutics; however, tools to quantify in situ drug exposure are few. We have investigated the potential of programmed death-ligand 1 (PD-L1) pharmacodynamics, quantified using PET, to inform on the tumor exposure of anti–PD-L1 (aPD-L1) therapeutics. Experimental Design: To noninvasively quantify PD-L1 levels, we first developed a novel peptide-based gallium-68–labeled binder, [68Ga]Ga-DK223, and evaluated its in vivo distribution, pharmacokinetics, and PD-L1 specificity in preclinical models of triple-negative breast cancer and urothelial carcinoma with variable PD-L1 expression. We then quantified baseline and accessible PD-L1 levels in tumors as a noninvasive pharmacodynamic measure to assess tumor exposure to two aPD-L1 antibodies (avelumab and durvalumab). Results: DK223 exhibited a KD of 1.01±0.83 nmol/L for PD-L1 and inhibited the PD-1:PD-L1 interaction in a dose-dependent manner. [68Ga]Ga-DK223 provides high-contrast PET images within 60 minutes of administration and detects PD-L1 in an expression-dependent manner in xenograft models. PD-L1 pharmacodynamics measured using [68Ga]Ga-DK223-PET revealed that avelumab and durvalumab had similar exposure early during therapy, but only durvalumab exhibited sustained exposure at the tumor. Conclusions: [68Ga]Ga-DK223 detected variable PD-L1 levels and exhibited salient features required for clinical translation. [68Ga]Ga-DK223-PET could be useful for quantifying total PD-L1 levels at baseline and accessible PD-L1 levels during therapy to understand drug exposure at the tumor, thus supporting its use for guiding and optimizing ICT.

Aedes aegypti acts as a vector for several arboviral diseases that impose a major socio-economic burden. Moreover, the absence of a vaccine against these diseases and drug resistance in mosquitoes necessitates the development of new control strategies for vector-borne diseases. ABC transporters that play a vital role in immunity and other cellular processes in different organisms may act as non-canonical immune molecules against arboviruses, however, their role in mosquito immunity remains unexplored. This study comprehensively analyzed various genetic features of putative ABC transporters and classified them into A-H subfamilies based on their evolutionary relationships. Existing RNA-sequencing data analysis indicated higher expression of cytosolic ABC transporter genes (E & F Subfamily) throughout the mosquito development, while members of other subfamilies exhibited tissue and time-specific expression. Furthermore, comparative gene expression analysis from the microarray dataset of mosquito infected with dengue, yellow fever and West Nile viruses revealed 31 commonly expressed ABC transporters suggesting a potentially conserved transcriptomic signature of arboviral infection. Among these, only a few transporters of ABCA, ABCC and ABCF subfamily were upregulated, while most were downregulated. This indicates the possible involvement of ABC transporters in mosquito immunity.

Background: Combination therapies that aim to improve the clinical efficacy to immune checkpoint inhibitors have led to the need for non-invasive and early pharmacodynamic biomarkers. Positron emission tomography (PET) is a promising non-invasive approach to monitoring target dynamics, and programmed death-ligand 1 (PD-L1) expression is a central component in cancer immunotherapy strategies. [¹⁸F]DK222, a peptide-based PD-L1 imaging agent, was investigated in this study using humanized mouse models to explore the relationship between PD-L1 expression and therapy-induced changes in cancer. Methods: Cell lines and xenografts derived from three non-small cell lung cancers (NSCLCs) and three urothelial carcinomas (UCs) were used to validate the specificity of [¹⁸F]DK222 for PD-L1. PET was used to quantify anti-programmed cell death protein-1 (PD-1) therapy-induced changes in PD-L1 expression in tumors with and without microsatellite instability (MSI) in humanized mice. Furthermore, [¹⁸F]DK222-PET was used to validate PD-L1 pharmacodynamics in the context of monotherapy and combination immunotherapy in humanized mice bearing A375 melanoma xenografts. PET measures of PD-L1 expression were used to establish a relationship between pathological and immunological changes. Lastly, spatial distribution analysis of [¹⁸F]DK222-PET was developed to assess the effects of different immunotherapy regimens on tumor heterogeneity. Results: [¹⁸F]DK222-PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high but variable tumor uptake at 60 min that correlated with PD-L1 expression. In MSI tumors treated with anti-PD-1, [¹⁸F]DK222 uptake was higher than in control tumors. Moreover, [¹⁸F]DK222 uptake was higher in A375 tumors treated with combination therapy compared with monotherapy, and negatively correlated with final tumor volumes. In addition, a higher number of PD-L1+ cells and higher CD8⁺-to-CD4⁺ cell ratio was observed with combination therapy compared with monotherapy, and positively correlated with PET. Furthermore, spatial distribution analysis showed higher [¹⁸F]DK222 uptake towards the core of the tumors in combination therapy, indicating a more robust and distinct pattern of immune cell infiltration. Conclusion: [¹⁸F]DK222-PET has potential as a non-invasive tool for monitoring the effects of immunotherapy on tumors. It was able to detect variable PD-L1 expression in tumors of different cancer types and quantify therapy-induced changes in tumors. Moreover, [¹⁸F]DK222-PET was able to differentiate the impact of different therapies on tumors.