Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans.

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.

Statistical and Methodological Considerations for Randomized Controlled Trial Design in Physical Medicine and Rehabilitation.

ABSTRACT: Well-designed randomized controlled clinical trials assessing treatments in the field of physical medicine and rehabilitation are essential for evidence-based patient care. However, there are challenges unique to clinical trials in physical medicine and rehabilitation due to complex health interventions in this field. We highlight routinely encountered empirical challenges and provide evidence-based recommendations on statistical and methodological approaches for the design and conduct of randomized controlled trials. Some of the issues addressed include challenges with blinding treatment groups in a rehabilitation setting, heterogeneity in treatment therapy, heterogeneity of treatment effects, uniformity in patient-reported outcome measures, and effect on power with varying scales of information. Furthermore, we discuss challenges with estimation of sample size and power, adaption to poor compliance with treatment and missing outcomes, and preferred statistical approaches for longitudinal data analysis. TO CLAIM CME CREDITS: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME. CME OBJECTIVES: Upon completion of this article, the reader should be able to: (1) Appraise the complexities of interventions in physical medicine and rehabilitation and how these challenges impact the conduct of clinical trials; (2) Develop an analytical strategy for poor treatment compliance and missing outcomes that can compromise the causal effect sought in a randomized clinical trial; and (3) Recognize the role of a data and safety monitoring board in a clinical trial. LEVEL: Advanced. ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s) ™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

Incidence and Clinical Risk Factors of Post-Operative Complications following Primary Total Hip Arthroplasty: A 10-Year Population-Based Cohort Study.

Background: Total hip arthroplasty (THA) has become a growing treatment procedure for debilitating hip pathologies. Patients experienced post-operative complications and revision surgeries according to large THA registries. To fully understand the short-term and long-term post-operative outcomes following THA, the purpose of this study is to examine the incidence of post-operative complications following primary THA and to examine how this trend has changed over 10 years within community hospitals in the US using large databases. Methods: This study queried the State Inpatient Database (SID) for primary THA between 2006 and 2015. Individual patients were followed forward in time until the first instance of a post-operative complication. The multivariable logistic regression analyses were computed to examine which post-operative complications were independent predictors of pre-operative comorbidities. Results: Median age of patients was 67 years, and 56% of patients were female. Females with avascular necrosis (AVN) as an indication for THA had a 27% higher risk of complication. Females with osteoarthritis (OA) as an indication for THA had a 6% higher risk of complication. Post-operative complications occurred with higher frequencies in the first two months of THA and the highest risks of THA complications within the first 6 months. Conclusion: The most common indication is OA in elders with primary THA. Females and those of black ethnicity showed the greatest risks of THA complications. Data from our large study can be used to understand post-operative complications and readmissions after THA. Our study also provides data on risk factors associated with these complications.

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.