Long-term spatiotemporal stability and dynamic changes in helminth infracommunities of spiny mice (Acomys dimidiatus) in St. Katherine’s Protectorate, Sinai, Egypt

The importance of parasites as a selective force in host evolution is a topic of current interest. However, short-term ecological studies of host-parasite systems, on which such studies are usually based, provide only snap-shots of what may be dynamic systems. We report here on four surveys, carried out over a period of 12 years, of helminths of spiny mice (Acomys dimidiatus), the numerically dominant rodents inhabiting the dry montane wadis in the Sinai Peninsula. With host age (age-dependent effects on prevalence and abundance were prominent) and sex (female bias in abundance in helminth diversity and in several taxa including Cestoda) taken into consideration, we focus on the relative importance of temporal and spatial effects on helminth infracommunities. We show that site of capture is the major determinant of prevalence and abundance of species (and higher taxa) contributing to helminth community structure, the only exceptions being Streptopharaus spp. and Dentostomella kuntzi. We provide evidence that most (notably the Spiruroidea, Protospirura muricola, Mastophorus muris and Gongylonema aegypti, but with exceptions among the Oxyuroidae e.g. Syphacia minuta), show elements of temporal-site stability, with rank order of measures among sites remaining similar over successive surveys and hence some elements of predictability in these systems

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen’s safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study’s A’herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

<div><p>HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent <i>in vitro</i> at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.</p></div

CD4 CARs respond to Env⁺ cells and not MHC class II⁺ cells.

<p><b>(A)</b> Primary human CD8 T cells were activated with either left NTD or transduced with the indicated CD4 CARs. Two weeks post activation, the CD8 T cells were co-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562 cells expressing high levels of HLA-DR, or K562 cells expressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression is shown on the left, and intracellular IL-2 expression and CD107a surface mobilization is shown on the right. <b>(B)</b> A co-culture assay was designed to demonstrate that CD4 CAR⁺ CD8 T cells do not kill MHC class II-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 T cells from <b>(A)</b> were co-cultured with K562 cells expressing both HLA-A2 and GFP as well as K562 expressing both HLA-DR*0401 and mCherry at a 1:1:1 ratio. Flow cytometry measuring GFP and mCherry expression was performed immediately after mixing (0 hr) and after 3 days of co-culture (72 hr). <b>C)</b> Summary data for a single experiment performed in triplicate, measuring the ratio of HLA-A2/GFP-expressing cells to HLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours of culture. Error bars indicate SEM. Data is representative of three independent experiments.</p

CAR T cells containing 4-1BB outperform CAR T cells containing CD28 in a humanized mouse HIV-treatment model.

<p>The experimental timeline and detailed description is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s008" target="_blank">S8 Fig</a>. CD4 T cell counts per μl of blood are shown (<b>A</b>) 3 days post ART initiation, prior to CD8 T cell injection (<b>B</b>) 18 days post ART removal and (<b>C</b>) at the endpoint termination bleeds (21 or 24 days post ART removal). For logistical reasons the mice had to be terminated in two groups, with BBz mice terminated 21 days post ART removal and the NTD and 28z terminated 24 days post ART removal. CD8 T cell counts are shown (<b>D</b>) 10 days post ART removal and CD8 T cell injection (<b>E</b>) 18 days post ART removal and (<b>F</b>) at the endpoint termination bleeds (21 or 24 days post ART removal). HIV RNA copies per μl plasma were determined by qPCR and normalized to CD4 T cell counts (<b>G</b>) 10 days post ART removal (<b>H</b>) 18 days post ART removal and (<b>I</b>) the endpoint bleed. The non-normalized viral loads are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s012" target="_blank">S12 Fig</a>. Mann Whitney Test was used to determine statistical significance (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001).</p

Lentiviral backbone augments CAR expression and control over HIV replication.

<p><b>(A-D)</b> Primary human CD8 T cells were activated with αCD3/αCD28 coated beads and were either left <b>(A)</b> nontransduced (NTD), <b>(B)</b> transduced with the original MMLV-based CD4 CAR, or <b>(C)</b> transduced with the same CAR placed in a HIV-based lentiviral vector, both driven by the PGK promoter. After eight days T cells were stained for CD4 and CD8 by flow cytometry. Median fluorescence intensity (MFI) is indicated on each graph. (<b>D)</b> Overlying histograms of the data shown in <b>(A-C). (E)</b> Eight days post activation, qPCR was performed and the number of integrated vector copies per cell was calculated. (<b>F</b>) Schematic of experimental design to study the control over HIV replication by T cells expressing HIV-specific CARs. Briefly, following activation with αCD3/αCD28 coated beads, CD4 T cells were infected with HIV Bal, and 24 hours later the indicated CD8 T cells were mixed at the indicated effector to target (E:T) ratios. After 7 days of co-culture, the expression of surface CD4, CD8, and intracellular Gag was measured by flow cytometry. <b>(G)</b> Intracellular Gag staining on CD8 negative cells, and <b>(H)</b> Intracellular Gag staining on CD8 positive cells. <b>(I)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate standard error of the mean (SEM). Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s014" target="_blank">S14 Fig</a> shows each of the 3 independent experiments. (<b>J)</b> Measurement of levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p