Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection

Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection

Exposure to Environmentally Relevant Concentrations of Genistein during Activation Does Not Affect Sperm Motility in the Fighting Fish Betta splendens

Sperm collected from male fighting fish Betta splendens were activated in control water, water containing the ion-channel blocker gadolinium (a putative positive control), or water containing the isoflavone phytoestrogen genistein to determine the effects of acute genistein exposure on male reproductive function. Computer-assisted sperm analysis was used to quantify the proportion of sperm that were motile and the swimming velocity of those sperm. The highest concentration of gadolinium (100 μM) tested was effective at reducing sperm motility and velocity, but neither concentration of genistein tested (3.7 nM or 3.7 μM) significantly affected these sperm parameters. Our findings suggest that acute exposure to waterborne phytoestrogens during activation does not reduce the motility of fish sperm

Journal Club: Anti‐CD19 Chimeric Antigen Receptor T Cell Therapy for Refractory Systemic Lupus Erythematosus

Objective Despite substantial advances in the treatment of systemic lupus erythematosus (SLE), some patients do not respond to the current state‐of‐the art therapies. This study assessed the tolerability and efficacy of CD19 chimeric antigen receptor (CAR) T cells in a small series of seriously ill and treatment‐resistant patients with SLE. Methods Five patients with SLE (four female patients and one male patient) with a median age of 22 (range 18–24) years, a median disease duration of 4 (range 1–9) years, and active disease (median Systemic Lupus Erythematosus Disease Activity Index score of 16 [range 8–16]) refractory to several immunosuppressive drug treatments were enrolled in a compassionate‐use CAR‐T cell program. Autologous T cells from patients with SLE were transduced with a lentiviral anti‐CD19 CAR vector, expanded, and reinfused at a dose of 1 × 106 CAR T cells per kilogram of body weight into the patients after lymphodepletion with fludarabine and cyclophosphamide. Results CAR T cells expanded in vivo and led to deep depletion of B cells, improvement of clinical symptoms, and normalization of laboratory parameters, including seroconversion of anti–double‐stranded DNA antibodies. Remission of SLE according to definition of remission in SLE criteria was achieved in all five patients after 3 months, and the median Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (range 2). Drug‐free remission was maintained during longer follow‐up (median of 8 [range 12] months after CAR‐T cell administration) and even after the reappearance of B cells, which was observed after a mean (±SD) of 110 ± 32 days after CAR‐T cell treatment. Reappearing B cells were naive and showed non–class‐switched B cell receptors. CAR‐T cell treatment was well tolerated, with only mild cytokine release syndrome. Conclusion These data suggest that CD19 CAR‐T cell therapy was feasible, tolerable, and effective in this small case series of refractory SLE. Nevertheless, larger placebo‐controlled trials are warranted

Data from: Mutational analysis of Rab3 function for controlling active zone protein composition at the Drosophila neuromuscular junction

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function

Human Immunodeficiency Virus Type 1 Pathobiology Studied in Humanized BALB/c-Rag2\u3csup\u3e-\u3c/sup\u3e/\u3csup\u3e-\u3c/sup\u3e γ\u3csub\u3ec\u3c/sub\u3e\u3csup\u3e-\u3c/sup\u3e/\u3csup\u3e-\u3c/sup\u3e Mice

The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34+ hematopoietic stem cells in BALB/c-Rag2-/- γc-/- mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5- coreceptor utilizing HIV-1ADA and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2 sup\u3e-/- γc-/- mice represent a unique and valuable resource for HIV-1 pathobiology studies

Q80L Rab3 Intensity

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

CDR Region Mutation Brp Distribution Rescue

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

Q80L Brp Distribution Rescue

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

Traces for Facilitation Index Quantification

Zipped folder contains .ABF Files. Explanation of files included as .txt file in Zipped Folde