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To explore induced islet neogenesis in the liver as a strategy for the treatment of diabetes, we used helper-dependent adenovirus (HDAD) to deliver the pancreatic duodenal homeobox-1 gene (Ipf1; also known as Pdx-1) to streptozotocin (STZ)-treated diabetic mice. HDAD is relatively nontoxic as it is devoid of genes encoding viral protein. Mice treated with HDAD-Ipf1 developed fulminant hepatitis, however, because of the exocrine-differentiating activity of Ipf1. The diabetes of STZ mice was partially reversed by HDAD-mediated transfer of NeuroD (Neurod), a factor downstream of Ipf1, and completely reversed by a combination of Neurod and betacellulin (Btc), without producing hepatitis. Treated mice were healthy and normoglycemic for the duration of the experiment ( [greater than] 120 d). We detected in the liver insulin and other islet-specific transcripts, including proinsulin-processing enzymes, [beta]-cell-specific glucokinase and sulfonylurea receptor. Immunocytochemistry detected the presence of insulin, glucagon, pancreatic polypeptide and somatostatin-producing cells organized into islet clusters; immuno-electron microscopy showed typical insulin-containing granules. Our data suggest that Neurod-Btc gene therapy is a promising regimen to induce islet neogenesis for the treatment of insulin-dependent diabetes., Author(s): Hideto Kojima [1]; Mineko Fujimiya [2]; Kazuhiro Matsumura [1]; Patrick Younan [1]; Hirotsugu Imaeda [2]; Makiko Maeda [1, 2]; Lawrence Chan (corresponding author) [1] Recent studies indicate that optimized [...]

Despite continued progress in the development of novel antiretroviral therapies, it has liecome increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV+ patients. In 2009, an HIV+ patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5-/-donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4+ T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4+ T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4+ T cells, an increase in nonmodified CD4+ T cells, enhanced cytotoxic. T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS. [ABSTRACT FROM AUTHOR]

Brentuximab vedotin, a CD30-directed antibody-drug conjugate (ADC), is approved for clinical use in multiple CD30-expressing lymphomas. The cytotoxic payload component of brentuximab vedotin is monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. Preclinical results provided here demonstrate that treatment of cancer cells with brentuximab vedotin or free MMAE leads to a catastrophic disruption of the microtubule network eliciting a robust endoplasmic reticulum (ER) stress response that culminates in the induction of the classic hallmarks of immunogenic cell death (ICD). In accordance with the induction of ICD, brentuximab vedotin-killed lymphoma cells drove innate immune cell activation in vitro and in vivo. In the "gold-standard" test of ICD, vaccination of mice with brentuximab vedotin or free MMAE-killed tumor cells protected animals from tumor rechallenge; in addition, T cells transferred from previously vaccinated animals slowed tumor growth in immunodeficient mice. Immunity acquired from killed tumor cell vaccination was further amplified by the addition of PD-1 blockade. In a humanized model of CD30+ B-cell tumors, treatment with brentuximab vedotin drove the expansion and recruitment of autologous Epstein-Barr virus-reactive CD8+ T cells potentiating the activity of anti-PD-1 therapy. Together, these data support the ability of brentuximab vedotin and MMAE to drive ICD in tumor cells resulting in the activation of antigen-presenting cells and augmented T-cell immunity. These data provide a strong rationale for the clinical combination of brentuximab vedotin and other MMAE-based ADCs with checkpoint inhibitors., (©2023 The Authors; Published by the American Association for Cancer Research.)

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4 + T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4 + T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4 + cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.

Unlabelled: Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4(+) T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4(+) T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4(+) T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4(+) T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4(+) T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4(+) T-lymphocyte loss, with at least 60% and 99% of CD4(+) T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival., Importance: HIV infection induces a massive depletion of CD4(+) T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4(+) T cells, uninfected CD4(+) T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The "bystander effects" that lead to enormous reductions in the number of uninfected CD4(+) T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV.

We investigated the role of intracellular calcium ([Ca(2+)](i)) in adhesion molecule expression in human umbilical vascular endothelial cells (HUVECs). Calmodulin (CaM) antagonists, W-7, trifluoperazine and chlorpromazine, triggered a rise in [Ca(2+)](i) in HUVECs. In the presence of extracellular Ca(2+), thapsigargin pretreatment completely prevented W-7-stimulated increase in [Ca(2+)](i), indicating that increase is attributable to the release of Ca(2+) from internal stores. The increased [Ca(2+)](i) acted as a second messenger to enhance tumor necrosis factor-alpha (TNF-alpha)-induced E-selectin and suppress intercellular cell adhesion molecule (ICAM-1) expression. Preincubation of HUVECs with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacetomethyl ester blocked W-7-mediated effects on E-selectin and ICAM-1. The W-7 effects were paralleled by changes in the respective mRNAs, suggesting regulation at a pretranslational level. These findings indicate that CaM-regulated [Ca(2+)](i) in HUVECs may play an important role in controlling expression of endothelial adhesion molecules involved in atherogenesis.