Primary Lymphocytes Research Articles

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN 2 as a regulator of effector functions

T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.

The immune-enhancing potential of peptide fractions from fermented Spirulina platensis by mixed probiotics.

Mixed fermentation with different microorganisms can facilitate fermentation metabolism and increase the low molecular metabolites accumulation, thereby enhancing the bioactive activity. In this study, we evaluated the immune-stimulating activities of Spirulina platensis and different extracts from fermented S. platensis (FS) by mixed probiotics in vitro, by measuring the proliferation and Th1/Th2 immunomodulatory potential on murine primary splenic lymphocytes. The results showed that mixed fermentation enhanced the immunomodulatory activity of S. platensis with higher lymphocyte proliferation compared with non-fermented S. platensis (NFS). Notably, the low molecular weight (<3kDa) peptide fraction from fermented S. platensis (L-PFS), especially at 40μg/ml, presented the strongest activity in promoting lymphocytes proliferation and modulating cytokines (IL-2 and IL-10) secretion. Meanwhile, L-PFS enhanced the relative mRNA expression of Th1 cytokine (IFN-γ) and Th2 cytokine (IL-4), along with inhibiting the relative mRNA expression of Th1 cytokines (IL-2 and TNF-α) and Th2 cytokine (IL-10) compared with Concanavalin A-treated lymphocytes. PRACTICAL APPLICATIONS: Fermentation with mixed probiotics could effectively improve the bioactive activity of S. platensis. In particular, L-PFS screened from the FS could significantly contribute to the immune-enhancing activity of lymphocytes, promote the Th1/Th2 balance, and provide insights for the investigation of FS as the potential immunomodulatory food products.

Integrative omics analyses uncover the mechanism underlying the immunotoxicity of perfluorooctanesulfonate in human lymphocytes

Perfluorooctanesulfonate (PFOS) is a man-made chemical widely used in industrial products. Due to its high persistence, PFOS has been detected in most animal species including the human population, wild animals, and aquatic organisms. Both cross-sectional studies and laboratory animal studies have shown hepatotoxicity, renal toxicity, and reproductive toxicity caused by PFOS exposure. Recently, a limited number of PFOS studies have raised concerns about its potential immune system effects. However, the molecular mechanism underlying the immunotoxicity of PFOS remains unknown. In this study, we used primary human lymphocytes as a model, together with integrative omics analyses, including the transcriptome and lipidome, and bioinformatics analysis, to resolve the immune toxicity effects of PFOS. Our results demonstrated that PFOS could alter the production of interleukins in human lymphocytes. Additionally, PFOS exposure could dysregulate clusters of genes and lipids that play important roles in immune functions, such as lymphocyte differentiation, inflammatory response, and immune response. The findings of this study offer novel insight into the molecular mechanisms underlying the immunotoxicity of PFOS, and open the potential of using the identified PFOS-responsive genes and lipids as biomarkers for risk assessment.

Decrease in immune function and the role of mitogen-activated protein kinase (MAPK) overactivation in apoptosis during T lymphocytes activation induced by zearalenone, deoxynivalenol, and their combinations

Currently there are few reports on the combined immunotoxicity of zearaleone (ZEA) and deoxynivalenol (DON). Since the two coexist naturally, it is necessary to understand the immunotoxicity caused by the two mycotoxins alone and in combination. To examine T lymphocytes activation and immune effect during activation, we used mouse primary spleen T lymphocytes as the experimental material and concanavalin (Con A) as the stimulator. The effects of ZEA, DON, and their combined exposure on T lymphocytes immune related function and the relationship between the activation of the mitogen-activated protein kinase (MAPK) signaling pathway and mycotoxin induced T lymphocytes apoptosis were studied in vitro. Specifically, T lymphocytes were isolated from primary mouse splenic lymphocytes, activated by Con A and then exposed to different concentrations of ZEA, DON, and their combinations. Our results showed that ZEA and DON alone and their combinations (20:1) can decrease the cell viability of T lymphocytes activated by Con A. The inhibitory effect of the combined groups was greater than that of the single mycotoxins, showing a synergistic effect. In addition, single or combined mycotoxins can lead to intracellular and surface ultrastructure damage of T lymphocytes, inhibit the expression of CD25 and CD278 and inhibit the synthesis of effect molecules poreforming protein (PFP), granzyme A (GZMA), and tumor necrosis factor-α (TNF-α). Meanwhile, the single mycotoxin or combined mycotoxins can promote the apoptosis of T lymphocytes which was accompanied by the overactivation of MAPK. After using the inhibitors of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) in the MAPK pathway, we found that the apoptosis of the cells induced by the ZEA was significantly decreased, and the apoptosis of the cells induced by DON had no significant changes. This suggests that the activation of MAPK induced by ZEA can promote the apoptosis of T lymphocytes, but the activation of MAPK induced by DON is not directly related to T cell apoptosis.

Lipoprotein(a) Cellular Uptake Ex Vivo and Hepatic Capture In Vivo Is Insensitive to PCSK9 Inhibition With Alirocumab

Lipoprotein(a) (Lp[a]) is the most common genetically inherited risk factor for cardiovascular disease. Many aspects of Lp(a) metabolism remain unknown. We assessed the uptake of fluorescent Lp(a) in primary human lymphocytes as well as Lp(a) hepatic capture in a mouse model in which endogenous hepatocytes have been ablated and replaced with human ones. Modulation of LDLR expression with the PCSK9 inhibitor alirocumab did not alter the cellular or the hepatic uptake of Lp(a), demonstrating that the LDL receptor is not a major route for Lp(a) plasma clearance. These results have clinical implications because they underpin why statins are not efficient at reducing Lp(a).

Design, antihuman immunodeficiency activity and molecular docking studies of synthesized 2-aryl and 2-pyrimidinyl pyrrolidines.

A series of thirty-one new compounds were synthesized and evaluated for their anti-HIV-1 and cytotoxicity activity. Of these, twelve were found to be inhibitors of HIV replications in primary human lymphocytes with median effective concentration (EC50) values < 20µM. However, most of the compounds demonstrated cytotoxicity in different cells. Our structure activity relationship study identified different patterns. In the series of 2-aryl pyrrolidines, comparing the activity of the compounds containing 2-aryl substituents we observed that compounds 1c, 1f-j, 2f,g with benzyloxyphenyl and isopropoxy groups were more potent. Compounds 1g-j, 2f,g, in which the 1-aryl moiety contained a methyl group in 3,5- or 4-positions also showed high activity. In the series of compounds containing the amide, aminomethyl and nitrile groups we observed an increase in activity with C(O)NH2 < CH2NH2 < CN. In the series of 2-pyrimidinyl pyrrolidines, the best results were demonstrated with derivatives 5e and 5f, in which the presence of a benzyl fragment in 1st and aniline fragment in 6th positions of pyrimidine ring we observed an increase in anti-HIV activity. Molecular docking studies of synthesized compounds with HIV-1 reverse transcriptase enzyme were performed. Binding energies of ligands were estimated, and the interacting amino acids of HIV-1 reverse transcriptase protein were shown. Based on corroborative results of the molecular docking studies and in vitro experiments, we suggest that three groups of synthesized ligands (1c, 1f-i), (2f,g), (5e,f, 7) are of high interest for further research on new drugs against HIV. General structure of synthesized 2-aryl and 2-pyrimidinyl pyrrolidines.

CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells

CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages invivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe invivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system.

CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells

Abstract CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.

CD8-Specific Designed Ankyrin Repeat Proteins Improve Selective Gene Delivery into Human and Primate T Lymphocytes.

Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo. This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Human ovarian cancer intrinsic mechanisms regulate lymphocyte activation in response to immune checkpoint blockade

Immune checkpoint blocking antibodies are currently being tested in ovarian cancer (OC) patients and have shown some responses in early clinical trials. However, it remains unclear how human OC cancer cells regulate lymphocyte activation in response to therapy. In this study, we have established and optimised an in vitro tumour-immune co-culture system (TICS), which is specifically designed to quantify the activation of multiple primary human lymphocyte subsets and human cancer cell killing in response to PD-1/L1 blockade. Human OC cell lines and treatment naïve patient ascites show differential effects on lymphocyte activation and respond differently to PD-1 blocking antibody nivolumab in TICS. Using paired OC cell lines established prior to and after chemotherapy relapse, our data reveal that the resistant cells express low levels of HLA and respond poorly to nivolumab, relative to the treatment naïve cells. In accordance, knockdown of IFNγ receptor expression compromises response to nivolumab in the treatment naïve OC cell line, while enhanced HLA expression induced by a DNA methyltransferase inhibitor promotes lymphocyte activation in TICS. Altogether, our results suggest a ‘cross resistance’ model, where the acquired chemotherapy resistance in cancer cells may confer resistance to immune checkpoint blockade therapy through down-regulation of antigen presentation machinery. As such, agents that can restore HLA expression may be a suitable combination partner for immunotherapy in chemotherapy-relapsed human ovarian cancer patients.

In vivo delivery of an exogenous molecule into murine T lymphocytes using a lymphatic drug delivery system combined with sonoporation

Physical delivery of exogenous molecules into lymphocytes is extremely challenging because conventional methods have notable limitations. Here, we evaluated the potential use of acoustic liposomes (ALs) and sonoporation to deliver exogenous molecules into lymphocytes within a lymph node (LN). MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice, which show systemic LN swelling, were used as the model system. After direct injection into the subiliac LN, a solution containing both ALs and TOTO-3 fluorophores (molecular weight: 1355) was able to reach the downstream proper axillary LN (PALN) via the lymphatic vessels (LVs). This led to the accumulation of a high concentration of TOTO-3 fluorophores and ALs in the lymphatic sinuses of the PALN, where a large number of lymphocytes were densely packed. Exposure of the PALN to >1.93 W/cm2 of 970-kHz ultrasound allowed the solution to extravasate into the parenchyma and reach the large number of lymphocytes in the sinuses. Flow cytometric analysis showed that TOTO-3 molecules were delivered into 0.49 ± 0.23% of CD8+7AAD− cytotoxic T lymphocytes. Furthermore, there was no evidence of tissue damage. Thus, direct administration of drugs into LVs combined with sonoporation can improve the delivery of exogenous molecules into primary lymphocytes. This technique could become a novel approach to immunotherapy.

Aberrant Migratory Behavior of Immune Cells in Recurrent Autoimmune Uveitis in Horses.

The participating signals and structures that enable primary immune cells migrating within dense tissues are not completely revealed until now. Especially in autoimmune diseases, mostly unknown mechanisms facilitate autoreactive immune cells to migrate to endogenous tissues, infiltrating and harming organ-specific structures. In order to gain deeper insights into the migratory behavior of primary autoreactive immune cells, we examined peripheral blood-derived lymphocytes (PBLs) of horses with equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis in humans. In this study, we used a three-dimensional collagen I hydrogel matrix and monitored live-cell migration of primary lymphocytes as a reaction to different chemoattractants such as fetal calf serum (FCS), cytokines interleukin-4 (IL-4), and interferon-γ (IFN-γ), and a specific uveitis autoantigen, cellular retinaldehyde binding protein (CRALBP). Through these experiments, we uncovered distinct differences between PBLs from ERU cases and PBLs from healthy animals, with significantly higher cell motility, cell speed, and straightness during migration of PBLs from ERU horses. Furthermore, we emphasized the significance of expression levels and cellular localization of septin 7, a membrane-interacting protein with decreased abundance in PBLs of autoimmune cases. To underline the importance of septin 7 expression changes and the possible contribution to migratory behavior in autoreactive immune cells, we used forchlorfenuron (FCF) as a reversible inhibitor of septin structures. FCF-treated cells showed more directed migration through dense tissue and revealed aberrant septin 7 and F-actin structures along with different protein distribution and translocalization of the latter, uncovered by immunochemistry. Hence, we propose that septin 7 and interacting molecules play a pivotal role in the organization and regulation of cell shaping and migration. With our findings, we contribute to gaining deeper insights into the migratory behavior and septin 7-dependent cytoskeletal reorganization of immune cells in organ-specific autoimmune diseases.

Anti-αLβ2 antibodies reveal novel endocytotic cross-modulatory functionality.

Antibodies targeting cell surface receptors are considered to enable highly selective therapeutic interventions for immune disorders and cancer. Their biological profiles are found, generally, to represent the net effects of antibody-target interactions. The former therapeutic anti-integrin αLβ2 antibody efalizumab seems to defeat this paradigm by eliciting, via mechanisms currently unknown, much broader effects than would be predicted based on its target specificity. To elucidate the mechanisms behind these broad effects, we investigated in primary human lymphocytes in vitro the effects of anti-αLβ2 antibodies on the expression of αLβ2 as well as unrelated α4 integrins, in comparison to Fab fragments and small-molecule inhibitors. We demonstrate that anti-αLβ2 mAbs directly induce the internalization of α4 integrins. The endocytotic phenomenon is a direct consequence of their antibody nature. It is inhibited when monovalent Fab fragments or small-molecule inhibitors are used. It is independent of crosslinking via anti-Fc mAbs and of αLβ2 activation. The cross-modulatory effect is unidirectional and not observed in a similar fashion with the α4 integrin antibody natalizumab. The present study identifies endocytotic cross-modulation as a hitherto unknown non-canonical functionality of anti-αLβ2 antibodies. This cross-modulation has the potential to fundamentally alter an antibody's benefit risk profile, as evident with efalizumab. The newly described phenomenon may be of relevance to other therapeutic antibodies targeting cluster-forming receptors. Thus, pharmacologists should be cognizant of this action when investigating such antibodies.

A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes.

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β7 integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.

Characterization and functional analysis of chicken APOBEC4

The APOBEC proteins play significant roles in the innate and adaptive immune system, probably due to their deaminase activities. Because APOBEC1 (A1) and APOBEC3 (A3) are absent in the chicken genome, we were interested in determining whether chicken APOBEC4 (A4) possessed more complex functions than its mammalian homologs. In this study, chicken A4 (chA4) mRNA was identified and cloned for the first time. Based on bioinformatics analyses, the conserved zinc-coordinating motif (HXE … PC(X)2–6C) was identified on the surface of chA4 and contained highly conserved His97, Glu99, Pro130, Cys131 and Cys138 active sites. The highest expression levels of constitutive chA4 were detected in primary lymphocytes and bursa of Fabricius. Newcastle Disease (ND) is one of the most serious infectious diseases in birds, causing major economic losses to the poultry industry. In vitro, Newcastle Disease Virus (NDV) early infection induced significant increases in chA4 expression in the chicken B cell line, DT40, the macrophage cell line, HD11 and the CD4+ T cell line, MSB-1, but not the fibroblast cell line, DF-1. In vivo, the expression levels of chA4 were up-regulated in several tissues from NDV-infected chickens, especially the thymus, testicles, duodenum and kidney. The high level expression of exogenous chA4 displayed inhibitory effects on NDV and reduced viral RNA in infected cells. Taken together, these data demonstrate that chA4 is involved in the chicken immune system and may play important roles in host anti-viral responses.

Down-regulation of the human major histocompatibility complex class I chain-related gene A (MICA) and its receptor is mediated by microRNA-146b-5p and is a potential mechanism of immunoediting in papillary thyroid carcinoma

Immune escape is one of the main reasons for the rapid progression of cancer and the poor efficacy of immunotherapy. Papillary thyroid cancer (PTC) is usually accompanied by intra-tumoral lymphocytic infiltration. The mechanisms regulating this tumor associated immune response or its evasion are not well understood. The major histocompatibility complex class I chain-related proteins A (MICA) and its receptor the natural killer group 2 member D (NKG2D) are major executers of the anti-tumor defense. This work aimed to study the expression and regulation of MICA-NKG2D and its association with the lymphocytic infiltration and miRNAs in PTC. Expression of MICA and NKG2D in thyroid tissues, and in cultured primary thyroid cancer cells and lymphocytes transfected with miR-146b-5p inhibitor/mimic was tested by RT-PCR. Results were confirmed by immunofluorescence staining and confocal microscopy. MICA is expressed in malignant and benign thyroid tissues with no association with aggressive behavior. Expression of MICA and NKG2D in PTC is concomitant with the presence of tumor associated lymphocytic response and is regulated by miR-146b-5p. MiR-146b-5p indirectly downregulates NKG2D expression in cancer cells and in lymphocytes. Overexpression of miR-146b-5p in PTC down-regulates MICA expression possibly to reduce the immunogenicity of the tumor cells. Targeting of the MICA-NKG2D axis by miR-146b-5p might be one of the ways adopted by thyroid cancer cells to aid the tumor in evading the immune response. The importance of our findings resides in the potential therapeutic use of MICA, NKG2D and miRNA-146b-5p as targets or modulators to enable the immune response against cancer.

The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/WAF1 in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21CIP1/WAF1, and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity.

Semen exosomes inhibit HIV infection and HIV-induced proinflammatory cytokine production independent of the activation state of primary lymphocytes.

Semen exosomes (SE) inhibit HIV infection. However, the effect of SE on cell activation and inflammation remains unknown. We characterized the response of peripheral blood mononuclear cells (PBMCs) from HIV-uninfected and antiretroviral therapy-suppressed HIV-infected (HIV+) subjects to SE. Quiescent PBMCs or T-cell receptor (TCR)-activated PBMCs from HIV- and HIV+ donors were stimulated with SE in the presence/absence of exvivo HIV infection. In HIV-infected PBMCs, SE did not reactivate HIV, did not induce lymphoblast development, nor increase CD69+/CD25+ numbers. Furthermore, SE inhibited denovo HIV infection without altering cell activation. SE also asynchronously downregulated HIV-inducible IL1β, IL8, and TNFα and upregulated CXCL10. These data suggest that SE inhibits HIV infection and production of HIV-induced proinflammatory cytokines while preserving lymphocyte activation.

Effect of massularia acuminata stem-bark extract on the mitochondrial oxygen consumption rate of cultured mouse astrocytes and primary lymphocytes

Effect of massularia acuminata stem-bark extract on the mitochondrial oxygen consumption rate of cultured mouse astrocytes and primary lymphocytes