Chimeric Approach Research Articles

Clinical trials for renal allograft tolerance induction through combined hematopoietic stem cell transplantation: A narrative review.

During the last four decades, development of effective immunosuppressants has significantly improved short-term results of organ transplantation. However, long-term results have not been satisfactory due to chronic rejection or complications caused by immunosuppressive drugs. Therefore, induction of immunological tolerance of the transplanted organ is considered essential to improve the long-term results. Despite numerous tolerance strategies that have been successful in murine models, inducing hematopoietic chimerism through donor hematopoietic stem cell transplantation is the only method that reproducibly induces kidney allograft tolerance in nonhuman primates or humans. Combining kidney and hematopoietic stem cell transplantation to achieve allograft tolerance has now been attempted with different chimerism strategies. This review summarizes the status of current clinical trials on the induction of allograft tolerance. We also summarize recent studies to extend the chimerism approach to deceased donor transplant recipients.

Monocyte proinflammatory phenotypic control by ephrin type A receptor 4 mediates neural tissue damage.

Circulating monocytes have emerged as key regulators of the neuroinflammatory milieu in a number of neuropathological disorders. Ephrin type A receptor 4 (Epha4) receptor tyrosine kinase, a prominent axon guidance molecule, has recently been implicated in the regulation of neuroinflammation. Using a mouse model of brain injury and a GFP BM chimeric approach, we found neuroprotection and a lack of significant motor deficits marked by reduced monocyte/macrophage cortical infiltration and an increased number of arginase-1+ cells in the absence of BM-derived Epha4. This was accompanied by a shift in monocyte gene profile from pro- to antiinflammatory that included increased Tek (Tie2 receptor) expression. Inhibition of Tie2 attenuated enhanced expression of M2-like genes in cultured Epha4-null monocytes/macrophages. In Epha4-BM–deficient mice, cortical-isolated GFP+ monocytes/macrophages displayed a phenotypic shift from a classical to an intermediate subtype, which displayed reduced Ly6chi concomitant with increased Ly6clo- and Tie2-expressing populations. Furthermore, clodronate liposome–mediated monocyte depletion mimicked these effects in WT mice but resulted in attenuation of phenotype in Epha4-BM–deficient mice. This demonstrates that monocyte polarization not overall recruitment dictates neural tissue damage. Thus, coordination of monocyte proinflammatory phenotypic state by Epha4 is a key regulatory step mediating brain injury.

Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera

SummaryBackgroundTo investigate a vaccine technology with potential to protect against coronavirus disease 2019 (COVID-19) and reduce transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a single vaccine dose, we developed a SARS-CoV-2 candidate vaccine using the live vesicular stomatitis virus (VSV) chimeric virus approach previously used to develop a licensed Ebola virus vaccine.MethodsWe generated a replication-competent chimeric VSV-SARS-CoV-2 vaccine candidate by replacing the VSV glycoprotein (G) gene with coding sequence for the SARS-CoV-2 Spike glycoprotein (S). Immunogenicity of the lead vaccine candidate (VSV∆G-SARS-CoV-2) was evaluated in cotton rats and golden Syrian hamsters, and protection from SARS-CoV-2 infection also was assessed in hamsters.FindingsVSV∆G-SARS-CoV-2 delivered with a single intramuscular (IM) injection was immunogenic in cotton rats and hamsters and protected hamsters from weight loss following SARS-CoV-2 challenge. When mucosal vaccination was evaluated, cotton rats did not respond to the vaccine, whereas mucosal administration of VSV∆G-SARS-CoV-2 was found to be more immunogenic than IM injection in hamsters and induced immunity that significantly reduced SARS-CoV-2 challenge virus loads in both lung and nasal tissues.InterpretationVSV∆G-SARS-CoV-2 delivered by IM injection or mucosal administration was immunogenic in golden Syrian hamsters, and both vaccination methods effectively protected the lung from SARS-CoV-2 infection. Hamsters vaccinated by mucosal application of VSV∆G-SARS-CoV-2 also developed immunity that controlled SARS-CoV-2 replication in nasal tissue.FundingThe study was funded by Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and The International AIDS Vaccine Initiative, Inc. (IAVI), New York, USA. Parts of this research was supported by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense.

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation.

The A3 adenosine receptor (A3AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure-activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo[4,5-c]quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A3AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A3AR signaling preferentially toward activation of Gαi3 and GαoA isoproteins, which are coexpressed with the A3AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A3AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A3AR PAMs, which includes leads for future pharmaceutical development.

Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2

SummaryBreakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

Dual Targeting Strategies on Histone Deacetylase 6 (HDAC6) and Heat Shock Protein 90 (Hsp90).

The design of multi-target drugs acting simultaneously on multiple signaling pathways is a growing field in medicinal chemistry, especially for the treatment of complex diseases, such as cancer. Histone deacetylase 6 (HDAC6) is an established anticancer drug target involved in tumor cells transformation. Being an epigenetic enzyme at the interplay of many biological processes, HDAC6 has become an attractive target for polypharmacology studies aimed at improving the therapeutic efficacy of anticancer drugs. For example, the molecular chaperone Heat shock protein 90 (Hsp90) is a substrate of HDAC6 deacetylation, and several lines of evidence demonstrate that simultaneous inhibition of HDAC6 and Hsp90 promotes synergistic antitumor effects on different cancer cell lines, highlighting the potential benefits of developing a single molecule endowed with multi-target activity. This review will summarize the complex interplay between HDAC6 and Hsp90, providing also useful hints for multi-target drug design and discovery approaches in this field. To this end, crystallographic structures of HDAC6 and Hsp90 complexes will be extensively reviewed in light of discussing binding pockets features and pharmacophore requirements and providing useful guidelines for the design of dual inhibitors. The few examples of multi-target inhibitors obtained so far, mostly based on chimeric approaches, will be summarized and put into context. Finally, the main features of HDAC6 and Hsp90 inhibitors will be compared, and ligand- and structure-based strategies potentially useful for the development of small molecular weight dual inhibitors will be proposed and discussed.

Cell Origin and iNOS Function Are Critical to Macrophage Activation Following Acute Lung Injury.

In the intratracheal bleomycin (ITB) model of acute lung injury (ALI), macrophages are recruited to the lung and participate in the inflammation and resolution that follows injury. Macrophage origin is influential in determining activation; however, the specific phenotype of recruited and resident macrophages is not known. Inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of ALI; however, the effects of its inhibition are mixed. Here we examined how macrophage origin determines the phenotypic response to ALI. Further, we hypothesize cell specific iNOS is key to determining activation and recruitment. Using a chimeric mouse approach, we have identified recruited and resident macrophage populations. We also used the chimeric mouse approach to create either pulmonary or bone marrow NOS2−/− mice and systemically inhibited iNOS via 1400 W. We evaluated macrophage populations at the peak of inflammation (8 days) and the beginning of resolution (15 days) following ITB. These studies demonstrate tissue resident macrophages adopt a M2 phenotype specifically, but monocyte originated macrophages activate along a spectrum. Additionally, we demonstrated that monocyte originating macrophage derived iNOS is responsible for recruitment to the lung during the inflammatory phase. Further, we show that macrophage activation is dependent upon cellular origin. Finally, these studies suggest pulmonary derived iNOS is detrimental to the lung following ITB. In conclusion, macrophage origin is a key determinant in response to ALI and iNOS is central to recruitment and activation.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Glutamate 73 Promotes Anti-arrhythmic Effects of Voltage-Dependent Anion Channel Through Regulation of Mitochondrial Ca2+ Uptake.

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.

Local FK506 implants in non-human primates to prevent early acute rejection in vascularized composite allografts.

BackgroundPrevious vascularized composite allograft (VCA) studies from our laboratory have shown that topical FK506 delivery in non-human primates (NHPs) was limited by inadequate dermal penetration and rejection persisted. Herein, we report the first utilization of FK506 via subcutaneously implanted discs to mitigate VCA rejection in NHPs.MethodsFull major histocompatibility complex (MHC)-mismatched NHP pairs underwent partial-face VCA and FK506 disc implantation along the suture line. All allotransplants were maintained post-operatively for two months on the FK506 discs, methylprednisolone, mycophenolate mofetil, and supplemented with intramuscular FK506 if necessary. Group 1 (n=4) was used for optimization of the implant, while Group 2 (n=3) underwent delayed bone marrow transplantation (DBMT) after two months. VCA skin biopsies and peripheral blood samples were obtained for serial assessment of rejection and mixed chimerism by histopathology and flow cytometry respectively.ResultsIn Group 1, two technical failures occurred. Of the remaining two NHPs, one developed supratherapeutic levels of FK506 (50–120 ng/mL) and had to be euthanized on postoperative day (POD) 12. Reformulation of the implant resulted in stable FK506 levels (20–30 ng/mL) up to POD12 when further intramuscular (IM) FK506 injections were necessitated. In Group 2, two NHPs survived to undergo conditioning and one successfully developed chimerism at 2–3 weeks post-DBMT (96–97% granulocytes and 7–11% lymphocytes of recipient-origin). However, all three NHPs had to be terminated from study at POD64, 77 and 86 due to underlying post-transplant lymphoproliferative disorder. All VCAs remained rejection-free up to study endpoint otherwise.ConclusionsThis study shows preliminary results of local FK506 implants in potentially mitigating VCA acute rejection for tolerance protocols based on mixed chimerism approach.

AML1/ETO and FLT3-ITD Cooperate to Induce Acute Myeloid Leukemia in Mice

Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, accounts for more than 40% of all leukemia deaths. The AML1-ETO (A) fusion gene resulting from t(8;21) is a recurring genetic alteration in human AML, typically associated with a favorable outcome. Internal tandem duplications (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (Flt3) result in constitutive ligand-independent signaling. Flt3ITD is one of the most frequently identified molecular abnormalities in AML, and when present, portends unfavorable clinical outcomes. AML1/ETO and FLT3ITD can co-exist in human AML, indicating that these lesions cooperate to promote disease development. Neither AML1/ETO nor Flt3ITD alone are sufficient to induce AML in mice. To determine whether coordinated expression of AML1/ETO and Flt3ITD would cause AML, Mx1-Cre x LSL AML1-ETO/+ x FLT3ITD/ITD (MAFF) mice were generated. In this system, an Mx1 promoter-driven Cre recombinase removes a lox-stop-lox (LSL) cassette upstream of a knock-in human AML1/ETO fusion gene in hematopoietic progenitor cells, while FLT3ITD is constitutively expressed. MAFF mice developed AML at 4-5 weeks of age with 100% penetrance. The AML was associated with severe leukocytosis, splenomegaly and dense bone marrow infiltration by immature-appearing blasts, and was universally lethal. Flow cytometry of bone marrow from MAFF mice revealed a striking expansion of cells harboring a hematopoietic stem cell (HSC) and multipotent progenitor (MP) cell phenotype, indicative of a block in normal myeloid differentiation. No lymphoid leukemias were observed. Interestingly, two copies of the FLT3ITD allele were required for AML to develop in this model. Transplantation of as few as 105 bone marrow cells from primary MAFF mice into lethally-irradiated secondary recipients resulted in 100% engraftment and lethality, with a leukemia identical to that which developed in primary MAFF mice, albeit with delayed latency. Mixed bone marrow chimeras were generated using MAFF marrow along with marrow from syngeneic, wild-type C57BL/6 or Rag2-/-γC-/- mice (lacking mature B, T and NK cells). Chimeras generated with MAFF and Rag2-/-γC-/- marrow succumbed more rapidly than those with MAFF and wild-type marrow, indicating that adaptive immunity and/or NK cells provided some level of active immune surveillance against MAFF AML. MAFF mice were crossed with Rosa26-LSLSIY/+ mice (referred to as MAFFS mice) in order to incorporate inducible expression of a tractable peptide antigen (SIYRYYGL; SIY) in leukemia cells. Through the use of mixed bone marrow chimeric approaches, we are now capable of monitoring SIY antigen-specific CD8+ T cell responses in MAFFS AML-bearing animals, including contexts in which the SIY antigen is shared (leukemia-associated antigen) or expressed only by leukemia cells (leukemia-specific antigen). In conclusion, we have generated the first autochthonous murine AML model in which the role of host immune surveillance can be interrogated. We expect that the MAFF AML model will also be useful for studies regarding leukemia biology and treatment. DisclosuresNo relevant conflicts of interest to declare.

TET-mediated regulation of effector CD4 T cells

Abstract Functionally distinct subsets of pathogen-specific CD4 helper T cells, including T follicular helper (Tfh) and T helper type 1 (Th1) subsets, are critical for orchestrating the induction and maturation of innate and adaptive immune responses. Proper genetic programming of Tfh and Th1 differentiation is required for regulating immune responses to clear primary infections and promote long-lived memory CD4 T cell-mediated protection against pathogen re-exposures. Genetic approaches to study CD4 T cell differentiation have been largely focused on transcriptional programming, but gene regulation is also critically tied to epigenetic modifications, which represents a major knowledge gap in our understanding of CD4 T cell biology. Ten-eleven-translocation (TET) family enzymes facilitate DNA demethylation to influence chromatin accessibility and promote gene expression. Prior work has shown that TET2 activity has been linked to optimal Th1 responses in vitro. TET1 and TET3 proteins mediate Cd4 locus demethylation and are required for stable CD4 expression during thymocyte development. However, the precise role of either TET1 or TET3 activity to mediate CD4 T cell fate and function in vivo has not been directly investigated. Thus, we hypothesized that TET1 and TET3 activity in peripheral, mature CD4 T cells is required for regulating T helper cell differentiation and function. We used conditional genetic and chimeric approaches, coupled with robust systems of infectious disease immunology, to directly test our hypothesis. Our data show substantial CD4 T cell developmental skewing towards Tfh fates in the absence of specific TET family members. This suggests that TET1 and TET3 enzymes play a role in governing Tfh and Th1 CD4 cell fate.

Characterization of a D1 Dopamine Receptor Positive Allosteric Modulator with a Novel Binding Site

The D1 dopamine receptor (D1R) is a crucial regulator of dopaminergic signaling and is involved in neurological processes and diseases. It is an attractive target for treating neuropsychiatric disorders, however, the liabilities of orthosteric agonists have curtailed this treatment modality. Positive allosteric modulators (PAMs) of D1R are therefore an alternative drug development strategy. We discovered two structurally distinct D1R PAMs via a high-throughput screen: MLS1082 and MLS6585. Both PAMs potentiate agonist-stimulated G protein- and β-arrestin-mediated signaling and increase dopamine's affinity for the D1R, though with different maximum efficacy and estimated Kb values. Combination experiments and receptor mutagenesis studies indicated that MLS1082 acts via the previously described intracellular loop-2 (ICL2) allosteric site targeted by two known D1R PAMs, Compound B and DETQ. MLS6585, however, does not act via this ICL2 site. To identify the MLS6585 binding site, chimeras of the D1R and D2R were used. MLS6585 has no PAM activity at the D2R, so loss of potentiation from the introduction of D2R sequences was used to detect potential regions of interest. This chimeric approach identified transmembrane region 7 (TM7) of D1R as a potential site for mediating MLS6585 activity. Further, specific point mutations identified residues near the extracellular region of TM7 that are required for MLS6585 PAM activity. These mutations had no effect on the activities of other PAMs binding to the ICL2 site. We used analog sets of MLS6585 to begin to understand the structure–activity relationships underlying D1R allosteric modulation. In addition to validating the MLS6585 scaffold as a D1R PAM, the analogs implicate structural moieties that are crucial for PAM activity and receptor selectivity. A small number of seemingly inactive analogs appear to act as silent allosteric modulators (SAMs) or neutral allosteric ligands (NALs), in that they blocked the activity of the parent PAM compound, presumably by competing for the same binding site. Together, these efforts increase our understanding of D1R allosteric modulation as a means for developing novel therapeutic interventions.

A Large-Scale Bank of Organ Donor Bone Marrow and Matched Mesenchymal Stem Cells for Promoting Immunomodulation and Transplant Tolerance.

Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed “hematopoietic progenitor cell (HPC), Marrow,” recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem (stromal) cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to “delayed tolerance.” Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT.

Mapping drug-target interactions and synergy in multi-molecular therapeutics for pressure-overload cardiac hypertrophy

Advancements in systems biology have resulted in the development of network pharmacology, leading to a paradigm shift from “one-target, one-drug” to “target-network, multi-component therapeutics”. We employ a chimeric approach involving in-vivo assays, gene expression analysis, cheminformatics, and network biology to deduce the regulatory actions of a multi-constituent Ayurvedic concoction, Amalaki Rasayana (AR) in animal models for its effect in pressure-overload cardiac hypertrophy. The proteomics analysis of in-vivo assays for Aorta Constricted and Biologically Aged rat models identify proteins expressed under each condition. Network analysis mapping protein–protein interactions and synergistic actions of AR using multi-component networks reveal drug targets such as ACADM, COX4I1, COX6B1, HBB, MYH14, and SLC25A4, as potential pharmacological co-targets for cardiac hypertrophy. Further, five out of eighteen AR constituents potentially target these proteins. We propose a distinct prospective strategy for the discovery of network pharmacological therapies and repositioning of existing drug molecules for treating pressure-overload cardiac hypertrophy.

The beta-glucuronidase intracisternal A particle insertion model results in similar overall MPSVII phenotype as the single base deletion model when on the same C57BL/6J mouse background

Two unique gene mutations in the enzyme beta-glucuronidase (GUSB) that result in the lysosomal storage disease Mucopolysaccharidosis (MPS) type VII had previously been reported to have differing disease phenotype severities when compared on differing mouse strains. The MPSVII mouse has proven to be a highly efficacious model to study mucopolysaccharidoses and for evaluating potential gene or stem cell therapies for lysosomal storage diseases. We examined the single base pair deletion (MPSVII) and the intracisternal A particle element insertion (MPSVII2J) in GUSB compared with control animals by skeletal measures, electroretinography, auditory-evoked brainstem response and life span on a C57BL/6J background strain. In all measures, both mutations result in either a trend toward or significant changes from the background strain control. In all measures, there is no significant phenotypic difference between the two mutations. The 2J variant is a more easily genotyped and equally affected phenotype, which holds promise for further studies of chimerism and stem cell therapy approaches.

Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests

DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.

Nanobodies as sensors of GPCR activation and signaling.

Nanobodies have emerged as useful tools to study G protein-coupled receptor (GPCR) structure, dynamic, and subcellular localization. Initially, several nanobodies have been developed as chaperones to facilitate GPCR crystallization. To explore their potential as biosensors to monitor receptor activation and dynamics, we here described protocols to characterize nanobody's interaction with GPCRs and their application as probes for protein identification and visualization on the cellular level. We also introduced a chimeric approach to enable a kappa-opioid receptor derived nanobody to bind to other GPCRs, including orphan GPCRs whose endogenous ligand or intracellular transducers are unknown. This approach provides a reporter assay to identify tool molecules to study the function of orphan GPCRs.

THE REQUIREMENT OF THYMIC IRRADIATION FOR INDUCTION OF MIXED CHIMERISM AND RENAL ALLOGRAFT TOLERANCE IN NON-HUMAN PRIMATES

Introduction: Thymic irradiation (TI) has been required for consistent induction of allograft tolerance through a mixed chimerism approach in both murine and nonhuman primate (NHP) models. In this study, we investigated the mechanisms of TI requirement in combined kidney (KTx) and bone marrow transplantation (BMT) in NHPs. Materials and Methods: In Group A, 5 recipients received BMT (combined KTx was evaluated in three) from MHC mismatched donors with a conditioning regimen that included total body irradiation (TBI, 1.5Gy), a Bcl-2 inhibitor (venetoclax), ATG and TI (7Gy). After transplant, the recipients were treated with a short course of anti-CD154 monoclonal antibody and a one-month course of cyclosporine. No immunosuppression was given after one month. In Group B, 3 recipients received combined KTx and BMT with the same regimen but without TI. Results: All recipients in Group A (TI) developed significantly higher chimerism than Group B (p < 0.02). Three recipients in Group A with KTx achieved long-term renal allograft survival without rejection (survival days 313, >646,>982 days) while all three Group B (No TI) recipients rejected renal allografts shortly after discontinuation of cyclosporine (97, 100, 163 days; p < 0.025 Groups A vs. B in Kaplan-Meier; Figure 1).Although recent thymic emigrants (RTE; CD4+CD31+CD45RA+) and naïve T cells of Group A were completely depleted after the preconditioning, those of Group B were substantially observed and significantly higher (Figure 2). On the other hand, no difference was observed in memory T cell subsets or regulatory T cells between two groups. Conclusion: Peripheral T cell deletion by the conditioning regimen may result in enhanced thymopoiesis with increased post-transplant RTE and naïve cells if TI is not administered. Higher post-transplant naïve T cells potentially inhibited chimerism and tolerance induction in recipients without TI.

Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis.

BackgroundVisceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL.Methodology/Principal findingsHere, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis.ConclusionThe newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.