Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na(+)-Cl(-) cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na(+) reabsorption and shifts Na(+) downstream for reabsorption by epithelial Na(+) channels (ENaC), which can drive K(+) secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K(+) concentration ([K(+)]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K(+)] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K(+)] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K(+), Na(+), and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na(+)-K(+)-2Cl(-) cotransporter (NKCC) phosphorylation 25% (P = 0.055). When plasma [K(+)] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na(+)/H(+) exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K(+) channel. In summary, raising plasma [K(+)] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K(+) ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na(+) reabsorption and flow drive K(+) secretion.

BST-2/tetherin is an interferon-inducible host restriction factor that blocks the release of newly formed enveloped viruses. It is enriched in lipid raft membrane microdomains, which are also the sites of assembly of several enveloped viruses. Viral anti-tetherin factors, such as the HIV-1 Vpu protein, typically act by removing tetherin from the cell surface. In contrast, the Ebola virus glycoprotein (GP) is unusual in that it blocks tetherin restriction without apparently altering its cell surface localization. We explored the possibility that GP acts to exclude tetherin from the specific sites of virus assembly without overtly removing it from the cell surface and that lipid raft exclusion is the mechanism involved. However, we found that neither GP nor Vpu had any effect on tetherin's distribution within lipid raft domains. Furthermore, GP did not prevent the colocalization of tetherin and budding viral particles. Contrary to previous reports, we also found no evidence that GP is itself a raft protein. Together, our data indicate that the exclusion of tetherin from lipid rafts is not the mechanism used by either HIV-1 Vpu or Ebola virus GP to counteract tetherin restriction.

Chimeric-antigen receptor (CAR)-T cell immunotherapies have been remarkably effective in treating acute lymphoblastic leukemia. However, current strategies generally suffer from difficult, inefficient and costly manufacturing processes, significant patient side effects and poor durability of response in some patients. Here, we report for the first time a CAR-T cell therapeutic comprising a non-immunoglobulin alternative scaffold Centyrin molecule (a "CARTyrin") manufactured with a novel non-viral piggyBacTM (PB) transposon-based system. Our lead candidate, P-BCMA-101, encodes a CARTyrin that targets the B cell maturation antigen (BCMA) for the treatment of multiple myeloma (MM) and has several unique aspects that improve upon earlier CAR-T products. First, P-BCMA-101 is manufactured using only in vitro transcribed mRNA and plasmid DNA without the need for lentivirus or g-retrovirus, resulting in time and cost savings. Importantly, PB is also safer than viral systems due to a less mutagenic insertional profile and is non-oncogenic. Furthermore, PB can efficiently deliver transgenes as large as several hundred kilobases, and, once inserted, transgenes demonstrate more stable, prolonged and higher expression when compared to those delivered by virus. Second, a mutein of the dihydrofolate reductase (DHFR) gene is included in the P-BCMA-101 transgene that can be used in combination with the non-genotoxic drug methotrexate (MTX) to provide a simple and effective method of CARTyrin+ cell enrichment and reduces variability in patient product material. Third, P-BCMA-101 incorporates a safety switch for optional depletion in vivo in case of adverse events. Lastly, the CARTyrin is comprised of a BCMA-specific Centyrin, which are based on a human tenascin fibronectin type III (FN3) consensus sequence. Centyrins have similar binding affinities to the antibody-derived single chain variable fragments (scFv), but are smaller, more thermostable and predicted to be less immunogenic. Importantly, no signs of tonic signaling leading to T cell exhaustion have been observed with CARTyrins unlike scFv-based CAR molecules, which can interact with each other on the surface causing non-specific CAR signaling. The manufacture process of P-BCMA-101 from primary human T cells is straightforward, employs no cytokines, and easily produces enough CARTyrin+ cells to treat patients. Within 18 days of electroporation of purified T cells, we demonstrate > 95% of the cell product is positive for CARTyrin expression and ready to be administered. Notably, our manufacturing process results in > 60% of CARTyrin+ T cells exhibiting a stem-cell memory phenotype (i.e. CD45RA+ CD62L+). P-BCMA-101 cells exhibit specific and robust in vitro activity against BCMA+ tumor targets, ranging from high to very low levels of BCMA, as measured by target-cell killing and CARTyrin-T cell proliferation. Importantly, proliferating P-BCMA-101 cells were highly sensitive in vitro to activation of the safety switch. Finally, we have evaluated the anti-tumor efficacy of P-BCMA-101 in a model of human MM. NSG™ mice were injected IV with 1.5x106 luciferase+ MM.1S cells, an aggressive human MM-derived cell line. After the tumor cells were allowed to grow for 21 days, animals received a single IV administration of 5x106 P-BCMA-101 cells. All untreated control animals demonstrated a marked increase in serum M-protein levels, rapid growth of tumor cells demonstrated by bioluminescent imaging (BLI), and death within four weeks. In stark contrast, 100% of animals that received P-BCMA-101 rapidly eliminated tumors within 7 days as measured by BLI and serum M-protein levels and improved survival out to at least 60 days post-treatment. P-BCMA-101 is the first-in-class of Centyrin-based CAR therapeutics. The CARTyrin, combined with our advanced manufacturing processes, represents a significant improvement over first generation, immunoglobulin-based and virally-transduced CAR-T products. P-BCMA-101 exhibited an advantageous stem-cell memory phenotype and demonstrated specific and potent anti-tumor efficacy against BCMA+ myeloma cells both in vitro and in vivo. Based on these results, we plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Disclosures Hermanson: Poseida Therapeutics: Employment. Barnett:Poseida Therapeutics: Employment. Rengarajan:Poseida Therapeutics: Employment. Codde:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

BackgroundThe interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity.ResultsBy examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin.ConclusionsThese results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

Background:Malnutrition and inflammation are associated with morbidity and mortality in patients on maintenance hemodialysis (MHD). Ghrelin, an orexigenic peptide hormone, is speculated to be associated with nutritional and inflammatory status in MHD.Aim:To assess the serum total ghrelin levels and its possible relationship with inflammation and nutritional status in patients on MHD.Subjects and Methods:The study was conducted on 90 patients on MHD for 6 months and above (56 males, 34 females, mean age 52.6 [11.7] years; mean dialysis vintage 20.9 [12.1] months) and 70 healthy volunteers as control (5 males, 25 females, mean age 50.6 [9.7] years). Demographics were obtained for the study population, and dialysis-related data were collected for cases. Anthropometry, biochemical parameters, serum total ghrelin and inflammatory markers tumor necrosis factor-alpha (TNF-α), and high-sensitivityC-reactive protein (hsCRP) were assessed for cases and control. Self-reported appetite (five questions of appetite and diet assessment tool) and nutritional status (subjective global assessment-dialysis malnutrition score) were assessed for cases.Results:Ghrelin (242.5 [62.3] pg/mL vs. 80.2 [19.6] pg/mL; P < 0.001), TNF-α (39.8 [15.2] pg/mL vs. 6.5 [1.2] pg/mL; P < 0.001), hsCRP (10.2 [2.8] mg/L vs. 2.7 [0.54] mg/L; P < 0.001) were significantly elevated in cases versus control, anthropometry, and biochemical parameters were significantly decreased in hemodialysis patient. Of 90 cases, (13/90 [14.4%]) were well-nourished, (28/90 [31%]) mild to moderately malnourished, and (49/90 [54.4%]) were moderate to severely malnourished. Appetite was very good for14.4%, good and fair for 47.8%, poor and very poor for 37.8% patients. There was a significant difference in appetite with respect to nutritional status (P < 0.001). Ghrelin had positive correlation with inflammatory markers and negative correlation with nutritional status (P < 0.001).Conclusion:The study identified the association of ghrelin with appetite, nutritional, and inflammatory status of the patients on MHD.