Generation of representative primary virus isolates from blood plasma after isolation of HIV-1 with CD44 MicroBeads

Infection of cell cultures with cell-free virus isolated from HIV-infected patients is notoriously difficult and results in a loss of viral variation. Here, we describe viral sequences from PBMC, U87.CD4.CCR5 and U87.CD4.CXCR4 cell cultures and compare them to those from blood plasma from 12 patients from whom virus particles were isolated using CD44 MicroBeads. In both PBMC and U87.CD4.CCR5 cultures, 66% of the plasma viral strains were retrieved after culturing. In addition, coreceptor use was predicted based on the env-V3 sequence and tested in U87.CD4 cells expressing either CCR5 or CXCR4. Recovery was lower for the CXCR4-using viruses. Only 50% of the virus clusters predicted to use CXCR4 could be retrieved from cell cultures, while 71% of CCR5-using strains were found in U87.CCR5 cultures. Therefore, isolation of primary viruses with CD44 MicroBeads results in a good representation in cell culture of the in vivo divergence

TREM-1 Protects HIV-1-Infected Macrophages from Apoptosis through Maintenance of Mitochondrial Function.

Macrophages are a reservoir for latent human immunodeficiency type 1 (HIV) infection and a barrier to HIV eradication. In contrast to CD4+ T cells, macrophages are resistant to the cytopathic effects of acute HIV infection. Emerging data suggest a role for TREM1 (triggering receptor expressed on myeloid cells 1) in this resistance to HIV-mediated cytopathogenesis. Here, we show that upon HIV infection, macrophages increase the expression of BCL2, BCLXL, TREM1, mitofusin 1 (MFN1), and MFN2 and the translocation of BCL2L11 (BIM) to the mitochondria and decrease the expression of BCL2-associated agonist of cell death (BAD) and BAX while maintaining a 95% survival rate over 28 days. The HIV proteins Tat and gp120 and the GU-rich single-stranded RNA (ssRNA) (RNA40) from the HIV long terminal repeat region (and a natural Toll-like receptor 8 [TLR8] agonist) induced similar effects. TREM1 silencing in HIV-infected macrophages led to decreased expression of BCL2, BCLXL, MFN1, and MFN2 and increased expression of BAD and BAX. This correlated with a significant increase in apoptosis mediated by a disruption of the mitochondrial membrane potential (Δψm), leading to the release of cytochrome c and caspase 9 cleavage. Exposure of TREM1-silenced macrophages to Tat, gp120, or RNA40 similarly resulted in the disruption of Δψm, cytochrome c release, caspase 9 cleavage, and apoptosis. Thus, our findings identify a mechanism whereby HIV promotes macrophage survival through TREM1-dependent upregulation of BCL2 family proteins and mitofusins that inhibits BCL2L11-mediated disruption of Δψm and subsequent apoptosis. These findings indicate that TREM1 can be a useful target for elimination of the HIV reservoir in macrophages.IMPORTANCE The major challenge to human immunodeficiency virus (HIV) treatment is the development of strategies that lead to viral eradication. A roadblock to accomplishing this goal is the lack of an approach that would safely eliminate HIV from all resting/latent reservoirs, including macrophages. Macrophages are a key part of the innate immune system and are responsible for recognizing invading microbes and sending appropriate signals to other immune cells. Here, we found that HIV induces the upregulation of the protein TREM1 (triggering receptor expressed on myeloid cells 1), which signals an increase in the expression of antiapoptotic proteins, thus promoting survival of HIV-infected macrophages

The Demise of Islet Allotransplantation in the US: A Call for an Urgent Regulatory Update The ISLETS FOR US Collaborative

Islet allotransplantation in the United States (US) is facing an imminent demise. Despite nearly three decades of progress in the field, an archaic regulatory framework has stymied US clinical practice. Current regulations do not reflect the state-of-the-art in clinical or technical practices. In the US, islets are considered biologic drugs and more than minimally manipulated human cell and tissue products (HCT/Ps). Across the world, human islets are appropriately defined as minimally manipulated tissue which has led to islet transplantation becoming a standard-of-care procedure for patients with type 1 diabetes mellitus and problematic hypoglycemia. As a result of the outdated US regulations, only eleven patients underwent allo-ITx in the US between 2011-2016 and all in the setting of a clinical trial. Herein, we describe the current regulations pertaining to islet transplantation in the United States. We explore the progress which has been made in the field and demonstrate why the regulatory framework must be updated to both, better reflect our current clinical practice and to deal with upcoming challenges. We propose specific updates to current regulations which are required for the renaissance of ethical, safe, effective, and affordable allo-ITx in the United States

Two HIV-1 Variants Resistant to Small Molecule CCR5 Inhibitors Differ in How They Use CCR5 for Entry

HIV-1 variants resistant to small molecule CCR5 inhibitors recognize the inhibitor-CCR5 complex, while also interacting with free CCR5. The most common genetic route to resistance involves sequence changes in the gp120 V3 region, a pathway followed when the primary isolate CC1/85 was cultured with the AD101 inhibitor in vitro, creating the CC101.19 resistant variant. However, the D1/86.16 escape mutant contains no V3 changes but has three substitutions in the gp41 fusion peptide. By using CCR5 point-mutants and gp120-targeting agents, we have investigated how infectious clonal viruses derived from the parental and both resistant isolates interact with CCR5. We conclude that the V3 sequence changes in CC101.19 cl.7 create a virus with an increased dependency on interactions with the CCR5 N-terminus. Elements of the CCR5 binding site associated with the V3 region and the CD4-induced (CD4i) epitope cluster in the gp120 bridging sheet are more exposed on the native Env complex of CC101.19 cl.7, which is sensitive to neutralization via these epitopes. However, D1/86.16 cl.23 does not have an increased dependency on the CCR5 N-terminus, and its CCR5 binding site has not become more exposed. How this virus interacts with the inhibitor-CCR5 complex remains to be understood

The demise of islet allotransplantation in the United States: A call for an urgent regulatory update

Islet allotransplantation in the United States (US) is facing an imminent demise. Despite nearly three decades of progress in the field, an archaic regulatory framework has stymied US clinical practice. Current regulations do not reflect the state-of-the-art in clinical or technical practices. In the US, islets are considered biologic drugs and “more than minimally manipulated” human cell and tissue products (HCT/Ps). In contrast, across the world, human islets are appropriately defined as “minimally manipulated tissue” and not regulated as a drug, which has led to islet allotransplantation (allo-ITx) becoming a standard-of-care procedure for selected patients with type 1 diabetes mellitus. This regulatory distinction impedes patient access to islets for transplantation in the US. As a result only 11 patients underwent allo-ITx in the US between 2016 and 2019, and all as investigational procedures in the settings of a clinical trials. Herein, we describe the current regulations pertaining to islet transplantation in the United States. We explore the progress which has been made in the field and demonstrate why the regulatory framework must be updated to both better reflect our current clinical practice and to deal with upcoming challenges. We propose specific updates to current regulations which are required for the renaissance of ethical, safe, effective, and affordable allo-ITx in the United States