Mechanisms of antigen-induced reversal of CNS inflammation in experimental demyelinating disease.

Autoimmune central nervous system (CNS) demyelinating diseases are a major public health burden and poorly controlled by current immunosuppressants. More precise immunotherapies with higher efficacy and fewer side effects are sought. We investigated the effectiveness and mechanism of an injectable myelin-based antigenic polyprotein MMPt (myelin oligodendrocyte glycoprotein, myelin basic protein and proteolipid protein, truncated). We find that it suppresses mouse experimental autoimmune encephalomyelitis without major side effects. MMPt induces rapid apoptosis of the encephalitogenic T cells and suppresses inflammation in the affected CNS. Intravital microscopy shows that MMPt is taken up by perivascular F4/80+ cells but not conventional antigen-presenting dendritic cells, B cells, or microglia. MMPt-stimulated F4/80+ cells induce reactive T cell immobilization and apoptosis in situ, resulting in reduced infiltration of inflammatory cells and chemokine production. Our study reveals alternative mechanisms that explain how cognate antigen suppresses CNS inflammation and may be applicable for effectively and safely treating demyelinating diseases.

BK virus-specific T cells for immunotherapy of progressive multifocal leukoencephalopathy: an open-label, single-cohort pilot study.

BACKGROUND: Progressive multifocal leukoencephalopathy, a rare disease of the CNS caused by JC virus and occurring in immunosuppressed people, is typically fatal unless adaptive immunity is restored. JC virus is a member of the human polyomavirus family and is closely related to the BK virus. We hypothesised that use of partly HLA-matched donor-derived BK virus-specific T cells for immunotherapy in progressive multifocal leukoencephalopathy would be feasible and safe. METHODS: We did an open-label, single-cohort pilot study in patients (aged 18 years or older) with clinically definite progressive multifocal leukoencephalopathy and disease progression in the previous month at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Overlapping peptide libraries derived from large T antigen and major capsid protein VP1 of BK virus with high sequence homology to JC virus counterparts were used to generate polyomavirus-specific T cells cross-recognising JC virus antigens. Polyomavirus-specific T cells were manufactured from peripheral blood mononuclear cells of first-degree relative donors aged 18 years or older. These cells were administered to patients by intravenous infusion at 1 × 106 polyomavirus-specific T cells per kg, followed by up to two additional infusions at 2 × 106 polyomavirus-specific T cells per kg. The primary endpoints were feasibility (no manufacturing failure based on meeting release criteria, achieving adequate numbers of cell product for clinical use, and showing measurable antiviral activity) and safety in all patients. The safety monitoring period was 28 days after each infusion. Patients were followed up with serial MRI for up to 12 months after the final infusion. This trial is registered at ClinicalTrials.gov, NCT02694783. FINDINGS: Between April 7, 2016, and Oct 19, 2018, 26 patients were screened, of whom 12 were confirmed eligible and received treatment derived from 14 matched donors. All administered polyomavirus-specific T cells met the release criteria and recognised cognate antigens in vitro. 12 patients received at least one infusion, ten received at least two, and seven received a total of three infusions. The median on-study follow-up was 109·5 days (range 23-699). All infusions were tolerated well, and no serious treatment-related adverse events were observed. Seven patients survived progressive multifocal leukoencephalopathy for longer than 1 year after the first infusion, whereas five died of progressive multifocal leukoencephalopathy within 3 months. INTERPRETATION: We showed that generation of polyomavirus-specific T cells from healthy related donors is feasible, and these cells can be safely used as an infusion for adoptive immunotherapy of progressive multifocal leukoencephalopathy. Although not powered to assess efficacy, our data provide additional support for this strategy as a potential life-saving therapy for some patients. FUNDING: Intramural Research Program of the National Institute of Neurological Disorders and Stroke of the NIH.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

In Situ Bioprinting of Autologous Skin Cells Accelerates Wound Healing of Extensive Excisional Full-Thickness Wounds.

The early treatment and rapid closure of acute or chronic wounds is essential for normal healing and prevention of hypertrophic scarring. The use of split thickness autografts is often limited by the availability of a suitable area of healthy donor skin to harvest. Cellular and non-cellular biological skin-equivalents are commonly used as an alternative treatment option for these patients, however these treatments usually involve multiple surgical procedures and associated with high costs of production and repeated wound treatment. Here we describe a novel design and a proof-of-concept validation of a mobile skin bioprinting system that provides rapid on-site management of extensive wounds. Integrated imaging technology facilitated the precise delivery of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into an injured area, replicating the layered skin structure. Excisional wounds bioprinted with layered autologous dermal fibroblasts and epidermal keratinocytes in a hydrogel carrier showed rapid wound closure, reduced contraction and accelerated re-epithelialization. These regenerated tissues had a dermal structure and composition similar to healthy skin, with extensive collagen deposition arranged in large, organized fibers, extensive mature vascular formation and proliferating keratinocytes.

Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications.

Bioprinting is an emerging technique used to fabricate viable, 3D tissue constructs through the precise deposition of cells and hydrogels in a layer-by-layer fashion. Despite the ability to mimic the native properties of tissue, printed 3D constructs that are composed of naturally-derived biomaterials still lack structural integrity and adequate mechanical properties for use in vivo, thus limiting their development for use in load-bearing tissue engineering applications, such as cartilage. Fabrication of viable constructs using a novel multi-head deposition system provides the ability to combine synthetic polymers, which have higher mechanical strength than natural materials, with the favorable environment for cell growth provided by traditional naturally-derived hydrogels. However, the complexity and high cost associated with constructing the required robotic system hamper the widespread application of this approach. Moreover, the scaffolds fabricated by these robotic systems often lack flexibility, which further restrict their applications. To address these limitations, advanced fabrication techniques are necessary to generate complex constructs with controlled architectures and adequate mechanical properties. In this study, we describe the construction of a hybrid inkjet printing/electrospinning system that can be used to fabricate viable tissues for cartilage tissue engineering applications. Electrospinning of polycaprolactone fibers was alternated with inkjet printing of rabbit elastic chondrocytes suspended in a fibrin-collagen hydrogel in order to fabricate a five-layer tissue construct of 1 mm thickness. The chondrocytes survived within the printed hybrid construct with more than 80% viability one week after printing. In addition, the cells proliferated and maintained their basic biological properties within the printed layered constructs. Furthermore, the fabricated constructs formed cartilage-like tissues both in vitro and in vivo as evidenced by the deposition of type II collagen and glycosaminoglycans. Moreover, the printed hybrid scaffolds demonstrated enhanced mechanical properties compared to printed alginate or fibrin-collagen gels alone. This study demonstrates the feasibility of constructing a hybrid inkjet printing system using off-the-shelf components to produce cartilage constructs with improved biological and mechanical properties.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Computational network model prediction of hemodynamic alterations due to arteriolar remodeling in interval sprint trained skeletal muscle.

OBJECTIVES: Exercise training is known to enhance skeletal muscle blood flow capacity, with high-intensity interval sprint training (IST) primarily affecting muscles with a high proportion of fast twitch glycolytic fibers. The objective of this study was to determine the relative contributions of new arteriole formation and lumenal arteriolar remodeling to enhanced flow capacity and the impact of these adaptations on local microvascular hemodynamics deep within the muscle. METHODS: The authors studied arteriolar adaptation in the white/mixed-fiber portion of gastrocnemius muscles of IST (6 bouts of running/day; 2.5 min/bout; 60 m/min speed; 15% grade; 4.5 min rest between bouts; 5 training days/wk; 10 wks total) and sedentary (SED) control rats using whole-muscle Microfil casts. Dimensional and topological data were then used to construct a series of computational hemodynamic network models that incorporated physiological red blood cell distributions and hematocrit and diameter dependent apparent viscosities. RESULTS: In comparison to SED controls, IST elicited a significant increase in arterioles/order in the 3A through 6A generations. Predicted IST and SED flows through the 2A generation agreed closely with in vivo measurements made in a previous study, illustrating the accuracy of the model. IST shifted the bulk of the pressure drop across the network from the 3As to the 4As and 5As, and flow capacity increased from 0.7 mL/min in SED to 1.5 mL/min in IST when a driving pressure of 80 mmHg was applied. CONCLUSIONS: The primary adaptation to IST is an increase in arterioles in the 3A through 6A generations, which, in turn, creates an approximate doubling of flow capacity and a deeper penetration of high pressure into the arteriolar network.