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

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems

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

ATV concentrations in plasma, lymph nodes, liver and spleen.

<p>Following intraperitoneal injection of nanoATV, plasma, mesenteric lymph nodes, liver and spleen were collected 1, 4, 12, 24, 48, and 72 h post injection. ATV concentrations were determined by UPLC-MS/MS. Tissue drug levels are reported as mean ± SEM for 4–8 mice/time point.</p

Identification of nanoATV in cells after intramuscular administration.

<p>Balb/c mice injected intramuscularly with 100 mg/kg CF633-nanoATV were sacrificed on days 1 and 7 after injection. Cryosections were prepared for H&E staining and for confocal microscopy. <b>(A)</b> Flow cytometric analysis of spleen demonstrates significant uptake of nanoATV in macrophages (CD45+CD3-CD11b+F4/80+) compared to lymphocytes (CD45+CD3+) and neutrophils/granulocytes (CD45+CD3-CD11b+F4/80-) at days 1 and 7. Data are represented as mean per mouse ± SEM and considered significant at P<0.05 (Student’s t-test). <b>(B)</b> Cryosections of muscle at the site of injection stained with H&E; 40X. <b>(C)</b> The muscle cryosection was stained with DAPI for nuclei (blue). The red arrowheads show the accumulation of nanoATV at the site of injection, 63X. <b>(D)</b> Macrophages were immunostained with CD68 antibody and Alexa Fluor 488 secondary antibody. Colocalization of nanoATV (pink) and CD68+ cells (green) at the site of injection is indicated with red arrowheads. Nuclei were stained with DAPI (blue), 63X. (scale bars = 10 μm)</p

Schematic of nanoATV cellular uptake and tissue distribution.

<p>Balb/c mice are injected with nanoATV directly into the peritoneum. This leads to an inflammatory reaction in which primarily granulocytes are induced. The nanoparticles are taken up, principally by macrophages and to a lesser degree by granulocytes. These cells and unincorporated nanoparticles leave the blood to reach Kupffer cells in the liver, splenic macrophages and other phagocytes including those in lymph nodes. In tissue the macrophages encase and store the drug particles in endosomal compartments.</p

Differential cell counts in peritoneal lavage.

<p>Following intraperitoneal injection of zymosan A, nanoATV, or P188 (control), cells were collected by peritoneal lavage at 1, 4, 12, 24, 48, and 72 h. Collected cells were stained with antibodies directed against GR-1, CD11b, and F4/80 and sorted by FACS. Mean total cell numbers per mouse ± SEM were calculated from 4–8 mice/treatment group/time point.</p

Identification of nanoATV within Kupffer cells.

<p>Confocal microscopy images of cryo-sectioned liver collected 12 h after intraperitoneal injection of nanoATV. Immunofluorescence images are as follows: <b>(A)</b> Cell nuclei stained with DAPI (blue); <b>(B)</b> Macrophages (Kupffer cells) stained with F4/80 and Alexa Fluor 488 (green); <b>(C)</b> NanoATV labeled with CF633 (pink); <b>(D)</b> Overlay of F4/80-labeled macrophages and CF633-labeled nanoATV. Images were acquired with a LSM 710 confocal microscope at 40x. (scale bars = 10 μm).</p