NK cells and monocytes modulate primary HTLV-1 infection

We investigated the impact of monocytes, NK cells, and CD8⁺ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1_WT) or to the HTLV-1_p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8⁺ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8⁺ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1_WT. In contrast, HTLV-1_p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1_WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1_p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1_WT and HTLV-1_p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 “don’t-eat-me” signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Changes in white oak (Quercus alba) phytochemistry in response to periodical cicadas: Before,during, and after an emergence

Periodical cicadas have mass emergences once every 13 or 17 years. Plants may need to upregulate defense production in response to an emergence. Defense production is energetically expensive, so plants may downregulate their production after periodical cicada populations dissipate. We examined the defensive responses in leaves, branches, and roots of a common host, white oak (Quercus alba), prior to, during, and after a 17‐year periodical cicada (Magicicada spp.) emergence in western Pennsylvania, United States. During the emergence, total tannins and condensed tannins increased in foliar tissue, while simultaneously decreasing in root tissue compared to the prior and subsequent years. Non‐structural carbohydrates were low prior to the mass emergence but were re‐allocated to belowground storage during the emergence year and dropped thereafter. In the year after the emergence, there was a relaxation of foliar defenses, and root defenses returned to pre‐emergence concentrations. We also tested for differences in damaged and undamaged branches on the same tree during (2019) and the year after the emergence (2020). Both damaged and undamaged branches had significantly greater chemical defenses (polyphenols, total tannins, and condensed tannins) during the emergence than in the following year when there was no emergence. We propose that re‐allocation of resources may help maximize oak tree fitness by moving resources away from areas that are not in immediate threat to areas that are under immediate threat. Changes in aboveground and belowground phytochemistry in response to periodical cicada mass emergences may help us better understand which resource re‐allocation strategies are used by plants to minimize the effects of insect emergencies.     

IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs allow simulation of fundamental hypotheses governing developmental programs and help accelerate the design of strategies to control stem cell fate. We first describe the architecture of IQCELL. Next, we apply IQCELL to scRNA-seq datasets from early mouse T-cell and red blood cell development, and show that the platform can infer overall over 74% of causal gene interactions previously reported from decades of research. We will also show that dynamic simulations of the generated GRN qualitatively recapitulate the effects of known gene perturbations. Finally, we implement an IQCELL gene selection pipeline that allows us to identify candidate genes, without prior knowledge. We demonstrate that GRN simulations based on the inferred set yield results similar to the original curated lists. In summary, the IQCELL platform offers a versatile tool to infer, simulate, and study executable GRNs in dynamic biological systems.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Altering Cellular Signaling Pathways Enhance Gene Silencing Activity of shRNA,shRNA.Ribozyme,and shRNA.Antisense in Neuroblastoma Cells

1. RNA interference (RNAi) is a multicomponent machinery that operates in a sequence-specific manner to repress the expression of genes in most eukaryotic cells.2. Here we wanted to investigate in a murine neuroblastoma cell line (NBP2) (a) if replacement of the loop of the short hairpin RNA (shRNA) with a hammerhead ribozyme (shRNA.RZ) or an antisense oligonucleotide (shRNA.AS) would affect the efficacy of gene suppression, and (b) if activation or inhibition of signaling pathways would enhance the efficacy of shRNA, shRNA.RZ, and shRNA.AS complex in gene silencing.3. We used U6-driven expression of these shRNAs to target either a short-lived green fluorescent protein (d2EGFP) or an endogenous cyclophilin A (CyP-A) gene in a d2EGFP expressing NBP2 cell line (NBP2-PN25).4. Activation of the cAMP signaling pathway or inhibition of phosphatidylinositol 3-kinase (PI3K) enhanced the efficacy of shRNA and shRNA.RZ complex in reducing the expression of d2EGFP shRNA.RZ complex was as efficacious as shRNA in reducing the expression of d2EGFP and CyP-A shRNA.AS complex showed a slightly lower efficacy than shRNA alone in decreasing d2EGFP expression. In contrast, the U6-driven hammerhead ribozyme targeted to d2EGFP showed no gene silencing activity.5. This report describes novel strategies of modifying shRNA and altering signaling pathways to affect siRNA-mediated gene silencing in a neuronal cell line.     

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer’s disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer’s disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.     

Anti-idiotype responses abrogate anti-CD4–induced tolerance to a tumor-specific antigen and promote systemic tumor immunity

Purpose: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. Methods: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. Results: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. Conclusions: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4⁺ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.Abbreviations CTL Cytotoxic T lymphocyte - FITC Fluorescein isothiocyanate - OD Optical density - PBS Phosphate-buffered saline - SV40 Simian virus 40