Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses.

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication.

Hunchback prevents notch-induced apoptosis in the serotonergic lineage of Drosophila Melanogaster.

The serotonergic lineage (NB7-3) in the Drosophila ventral nerve cord produces six cells during neurogenesis. Four of the cells differentiate into neurons: EW1, EW2, EW3 and GW. The other two cells undergo apoptosis. This simple lineage provides an opportunity to examine genes that are required to induce or repress apoptosis during cell specification. Previous studies have shown that Notch signaling induces apoptosis within the NB7-3 lineage. The three EW neurons are protected from Notch-induced apoptosis by asymmetric distribution of Numb protein, an inhibitor of Notch signaling. In a numb1 mutant EW2 and EW3 undergo apoptosis. The EW1 and GW neurons survive even in a numb1 mutant background suggesting that these cells are protected from Notch-induced apoptosis by some factor other than Numb. The EW1 and GW neurons are mitotic sister cells, and uniquely express the transcription factor Hunchback. We present evidence that Hunchback prevents apoptosis in the NB7-3 lineage during normal CNS development and can rescue the two apoptotic cells in the lineage when it is ectopically expressed. We show that hunchback overexpression produces ectopic cells that express markers similar to the EW2 neuron and changes the expression pattern of the EW3 neuron to a EW2 neuron. In addition we show that hunchback overexpression can override apoptosis that is genetically induced by the pro-apoptotic genes grim and hid.

Active Postlicensure Safety Surveillance for Recombinant Zoster Vaccine Using Electronic Health Record Data.

Recombinant zoster vaccine (RZV) (Shingrix; GlaxoSmithKline, Brentford, United Kingdom) is an adjuvanted glycoprotein vaccine that was licensed in 2017 to prevent herpes zoster (shingles) and its complications in older adults. In this prospective, postlicensure Vaccine Safety Datalink study using electronic health records, we sequentially monitored a real-world population of adults aged ≥50 years who received care in multiple US Vaccine Safety Datalink health systems to identify potentially increased risks of 10 prespecified health outcomes, including stroke, anaphylaxis, and Guillain-Barré syndrome (GBS). Among 647,833 RZV doses administered from January 2018 through December 2019, we did not detect a sustained increased risk of any monitored outcome for RZV recipients relative to either historical (2013-2017) recipients of zoster vaccine live, a live attenuated virus vaccine (Zostavax; Merck & Co., Inc., Kenilworth, New Jersey), or contemporary non-RZV vaccine recipients who had an annual well-person visit during the 2018-2019 study period. We confirmed prelicensure trial findings of increased risks of systemic and local reactions following RZV. Our study provides additional reassurance about the overall safety of RZV. Despite a large sample, uncertainty remains regarding potential associations with GBS due to the limited number of confirmed GBS cases that were observed.

Incomplete reproductive barriers and genomic differentiation impact the spread of resistance mutations between green- and red-colour morphs of a cosmopolitan mite pest.

Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is imperative to develop sustainable crop management strategies. The two-spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green- or a red-coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso-female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on-going gene flow between green-coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target-site resistance mutations. Our results indicate that target-site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations.

An unusual case of checkpoint-inhibitor-induced pleuropericarditis.

INTRODUCTION: Pembrolizumab is an immune checkpoint inhibitor that promotes effector T-cell functions on malignant cells by binding to programmed cell death protein 1 (PD-1). Pembrolizumab is well tolerated in most cases with an adverse event profile consisting mainly of pruritus, fatigue, and anorexia. Cardiotoxicity comprises 1% of the total adverse events. CASE REPORT: We present a case of a 64-year-old female with non-small cell lung cancer (NSCLC) who developed pleuropericarditis following pembrolizumab therapy. MANAGEMENT & OUTCOME: The patient was successfully managed with colchicine, furosemide, and timely initiation of methylprednisolone with the improvement of her symptoms. The decision to discontinue pembrolizumab was made, and six months after this intervention, the patient has remained asymptomatic. DISCUSSION: Clinicians should recognize these potential immune-mediated adverse effects to provide effective and timely management and optimize patient care.

Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Use of Cells, Supplements, and Peptides as Therapeutic Strategies for Modulating Inflammation after Spinal Cord Injury: An Update.

Spinal cord injury is a traumatic lesion that causes a catastrophic condition in patients, resulting in neuronal deficit and loss of motor and sensory function. That loss is caused by secondary injury events following mechanical damage, which results in cell death. One of the most important events is inflammation, which activates molecules like proinflammatory cytokines (IL-1ß, IFN-γ, and TNF-α) that provoke a toxic environment, inhibiting axonal growth and exacerbating CNS damage. As there is no effective treatment, one of the developed therapies is neuroprotection of the tissue to preserve healthy tissue. Among the strategies that have been developed are the use of cell therapy, the use of peptides, and molecules or supplements that have been shown to favor an anti-inflammatory environment that helps to preserve tissue and cells at the site of injury, thus favoring axonal growth and improved locomotor function. In this review, we will explain some of these strategies used in different animal models of spinal cord injury, their activity as modulators of the immune system, and the benefits they have shown.

Polypropylene Nanocomposites Attained by In Situ Polymerization Using SBA-15 Particles as Support for Metallocene Catalysts: Effect of Molecular Weight and Tacticity on Crystalline Details, Phase Transitions and Rheological Behavior.

In this study, nanocomposites based on polypropylene are synthesized by the in situ polymerization of propene in the presence of mesoporous SBA-15 silica, which acts as a carrier of the catalytic system (zirconocene as catalyst and methylaluminoxane as cocatalyst). The protocol for the immobilization and attainment of hybrid SBA-15 particles involves a pre-stage of contact between the catalyst with cocatalyst before their final functionalization. Two zirconocene catalysts are tested in order to attain materials with different microstructural characteristics, molar masses and regioregularities of chains. Some polypropylene chains are able to be accommodated within the silica mesostructure of these composites. Thus, an endothermic event of small intensity appears during heating calorimetric experiments at approximately 105 °C. The existence of these polypropylene crystals, confined within the nanometric channels of silica, is corroborated by SAXS measurements obtained via the change in the intensity and position of the first-order diffraction of SBA-15. The incorporation of silica also has a very significant effect on the rheological response of the resultant materials, leading to important variations in various magnitudes, such as the shear storage modulus, viscosity and δ angle, when a comparison is established with the corresponding neat iPP matrices. Rheological percolation is reached, thus demonstrating the role of SBA-15 particles as filler, in addition to the supporting role that they exert during the polymerizations.

Effect of Mexico's vaccination program on Covid-19 cases, hospitalizations, and deaths among older adults in Mexico City.

OBJECTIVE: To characterize the impact of Mexico's Co-vid-19 vaccination campaign of older adults. MATERIALS AND METHODS: We estimated the absolute change in sympto-matic cases, hospitalizations and deaths for vaccine-eligible adults (aged >60 years) and the relative change compared to vaccine-ineligible groups since the campaign started. Re-sults. By May 3, 2021, the odds of Covid-19 cases among adults over 60 compared to 50-59 year olds decreased by 60.3% (95%CI: 53.1, 66.9), and 2 003 cases (95%CI: 1 156, 3 130) were avoided. Hospitalizations and deaths showed similar trends. CONCLUSIONS: Covid-19 events decreased after vaccine rollout among those eligible for vaccination.

Colectomy rate in ulcerative colitis 15 years after diagnosis: Results from the 2001-2003 Navarra cohort. / Tasas de colectomía en colitis ulcerosa 15 años después del diagnóstico. Datos de la cohorte Navarra 2001-2003.

INTRODUCTION: Knowing the natural history of ulcerative colitis (UC) is essential to understand the course of the disease, assess the impact of different treatment strategies and identify poor prognostic factors. One of the most significant matters in this regard is the need for surgery. OBJECTIVES: To analyse the Colectomy Incidence Rate (CIR) from diagnosis to end of follow-up (31/12/2017) and identify predictive factors for colectomy. MATERIAL AND METHODS: A retrospective study enrolling patients with a definitive diagnosis (DD) of UC or Unclassified Colitis (UnC) in the 2001-03 Navarra cohort. RESULTS: We enrolled 174 patients with a DD of UC (E2 42.8%; E3 26.6%) and 5 patients with a DD of UnC: 44.1% women, median age 39.2 years (range 7-88) and median follow-up 15.7 years. A total of 8 patients underwent surgery (CIR 3 colectomies/103 patient-years: 3 at initial diagnosis (<1 month), 2 in the first 2 years, 2 at 5 years from diagnosis and 1 at 12 years from diagnosis. All had previously received steroids; 5 had received immunomodulators and 2 had received biologics. In 7 patients (87%), surgery was performed on an emergency basis. The indication was megacolon in 3 (37.5%), severe flare-up in 3 (37.5%) and medical treatment failure in 2 (25%). In 5 cases (62.5%), an ileoanal pouch was made, and in 3 cases, a definitive ileostomy was performed. In the univariate analysis, patients with loss of more than 5 kg at diagnosis and admission at diagnosis had a lower rate of colectomy-free survival. CONCLUSIONS: In our series, colectomy rates are lower than usually reported. Most colectomies were performed in the first 5 years following diagnosis and had an emergency indication.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

The regulatory role of ProBDNF in monocyte function: Implications in Stanford type-A aortic dissection disease.

Brain-derived neurotrophic factor precursor (proBDNF) has been reported to strengthen the dysfunction of monocytes/macrophages in animal studies. However, it is still unknown the roles of proBDNF in the dysfunction of monocytes in the inflammatory diseases in humans. In the present study, we showed that proBDNF and pan neurotrophic receptor p75 were significantly upregulated in monocytes from healthy donors (HD) after lipopolysaccharide treatment. Exogenous proBDNF treatment upregulated CD40 and proinflammatory cytokines expression in monocytes including interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. In Stanford type-A acute aortic dissection (AAD) patients, proBDNF was upregulated in CD14+ CD163+ CX3CR1+ M2- but not CD14+ CD68+ CCR2+ M1-like monocytes. In addition, sera from AAD patients activated gene expression of proinflammatory cytokines in cultured PBMCs from HD, which was attenuated by proBDNF monoclonal antibody (Ab-proB) treatment. These findings suggested that upregulation of proBDNF in M2-like monocytes may contribute to the proinflammatory response in the AAD.

Performance characteristics of a fast real-time PCR assay for hepatitis B virus DNA quantification.

Detection of hepatitis B virus (HBV) DNA is particularly important for detection of early acute and of occult HBV infection. On the other hand, HBV DNA detection and quantification are essential to diagnose and treat chronic HBV infection. In this study, we evaluated the performance of the real-time PCR SUMASIGNAL VHB (un paso) (Immunoassay Center, Cuba). The clinical and analytical specificity of the assay was 100%. Intra-assay and inter-assay coefficients of variation ranged from 0.50 to 2.53% and from 1.23 to 3.03%, respectively. A strong correlation (r=0.926; P<0.001) with the COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, v2.0 (Roche Molecular Systems, Inc.) was obtained. The limit of detection using the third WHO international standard for HBV DNA was 377.47 IU/mL. The test was able to detect the most prevalent HBV genotypes (A-G) equally well. In conclusion, the SUMASIGNAL VHB (un paso) is a sensitive, specific, precise and accurate assay for the quantification of serum and plasma HBV DNA. Thus, this simple and fast real-time PCR test can be used as an aid in diagnosing an HBV infection and monitoring drug efficacy.

Racial disparities in prostate cancer survival in a screened population: Reality versus artifact.

BACKGROUND: Racial disparities in prostate cancer survival (PCS) narrowed during the prostate-specific antigen (PSA) era, suggesting that screening may induce more equitable outcomes. However, the effects of lead time and overdiagnosis can inflate survival even without real screening benefit. METHODS: A simulation model of PCS in the early PSA era (1991-2000) was created. The modeled survival started with baseline survival in the pre-PSA era (1975-1990) and added lead times and overdiagnosis using estimates from published studies. The authors quantified 1) discrepancies between modeled and observed PCS in the PSA era and 2) residual period effects on PCS given specified values for screening benefit. RESULTS: Lead time and overdiagnosis explained more of the improvement in PCS for older ages at diagnosis (46% [95% confidence interval (CI), 44%-50%] for blacks and 51% [95% CI, 50%-52%] for all races ages 50-54 years vs 98% [95% CI, 97%-99%] for blacks and 100% for all races ages 75-79 years). They also explained more of the narrowing in PCS disparities for older ages (33% [95% CI, 31%-43%] for men ages 50-54 years vs 74% [95% CI, 71%-81%] for men ages 75-79 years). The period effects amounted to reductions of 27% to 40% among blacks and 26% to 38% among all races in the risk of prostate cancer death, depending on the screening benefit. CONCLUSIONS: Real improvements in survival disparities in the PSA era are smaller than those observed and reflect similar reductions in the risk of prostate cancer death among blacks and all races. Understanding screening artifacts is necessary for valid interpretation of observed survival trends. Cancer 2018;124:1752-9. © 2018 American Cancer Society.

Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response.

Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-κB pathway inhibitors A52, B15, and K7 in immunity, we deleted their genes in an NYVAC (New York vaccinia virus) strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of the A52R, B15R, and K7R genes increased dendritic cell, natural killer cell, and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses with these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double-, and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly.IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of the central host-cell signaling pathway. Using mice, we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design.

Expression of Early Growth Response Gene-2 and Regulated Cytokines Correlates with Recovery from Guillain-Barré Syndrome.

Guillain-Barré syndrome (GBS) is an immune-mediated peripheral neuropathy. The goal of this research was the identification of biomarkers associated with recovery from GBS. In this study, we compared the transcriptome of PBMCs from a GBS patient and her healthy twin to discover possible correlates of disease progression and recovery. The study was then extended using GBS and spinal cord injury unrelated patients with similar medications and healthy individuals. The early growth response gene-2 (EGR2) was upregulated in GBS patients during disease recovery. The results provided evidence for the implication of EGR2 in GBS and suggested a role for EGR2 in the regulation of IL-17, IL-22, IL-28A, and TNF-ß cytokines in GBS patients. These results identified biomarkers associated with GBS recovery and suggested that EGR2 overexpression has a pivotal role in the downregulation of cytokines implicated in the pathophysiology of this acute neuropathy.

Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy.

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I-II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds. Graphical abstract Synchrotron IR microspectroscopy can provide insight into the response of neural progenitor cells to synthetic scaffolds.

NFκB activation by modified vaccinia virus as a novel strategy to enhance neutrophil migration and HIV-specific T-cell responses.

Neutrophils are antigen-transporting cells that generate vaccinia virus (VACV)-specific T-cell responses, yet how VACV modulates neutrophil recruitment and its significance in the immune response are unknown. We generated an attenuated VACV strain that expresses HIV-1 clade C antigens but lacks three specific viral genes (A52R, K7R, and B15R). We found that these genes act together to inhibit the NFκB signaling pathway. Triple ablation in modified virus restored NFκB function in macrophages. After virus infection of mice, NFκB pathway activation led to expression of several cytokines/chemokines that increased the migration of neutrophil populations (Nα and Nß) to the infection site. Nß cells displayed features of antigen-presenting cells and activated virus-specific CD8 T cells. Enhanced neutrophil trafficking to the infection site correlated with an increased T-cell response to HIV vector-delivered antigens. These results identify a mechanism for poxvirus-induced immune response and alternatives for vaccine vector design.

Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation.

BACKGROUND: ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer. RESULTS: Here, we show that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development. CONCLUSION: In addition to a role of dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP.