Determination of the Expression Patterns of Bovine Non-Classical Major Histocompatibility Complex (MHC) Class I Proteins

My dissertation hypothesis is that bovine trophoblast cells express cell-surface and secreted non-classical major histocompatibility complex class I (MHC-Ib) proteins which inhibit NK cells and other leukocytes by binding to inhibitory receptors (e.g., LILRB1, LILRB2, KIR2DL4, and/or CD94/NKG2A). Extremely polymorphic and ubiquitously expressed classical MHC class I (MHC-Ia) proteins, which present foreign antigenic peptides to CD8+ T lymphocytes, are involved in acceptance or rejection of tissue grafts. Non-classical MHC class I (MHC-Ib) glycoproteins, such as Human Leukocyte Antigen-G (HLA-G) and murine Qa-2, are important modulators of the maternal immune system during pregnancy. MHC-Ib proteins are: (a) oligomorphic or monomorphic, (b) expressed in specific tissues under specific condtions, and (c) produced as surface and/or soluble isoforms due to alternative splicing. Third trimester-bovine trophoblast cells express both MHC-Ia and MHC-Ib proteins. The MHC-Ib proteins expressed by trophoblast cells during the third trimester of pregnancy are encoded by four bovine leukocyte antigen (BoLA) loci: BoLA-NC1, BoLA-NC2, BoLA-NC3, and BoLA-NC4. Two MHC-Ia (N*01701 and N*01802) and three MHC-Ib (NC1*00501, NC3*00101 and NC4*00201) proteins showed cell-surface expression in transfection studies performed in murine P815 and human K562 cells. Two additional isoforms, NC1*00401 and NC2*00102, were not detected on the surface of these cells. Nevertheless, both class Ia proteins, N*01701 and N*01802, and five class Ib proteins, NC1*00401, NC1*00501, NC2*00102, NC3*00101, and NC4*00201, were detected in crude cell lysates on Western blots. Precipitation of proteins from culture supernatants showed that cell-surface MHC-Ia (N*01701 and N*01802) and MHC-Ib proteins (NC1*00501, NC3*00101, and NC4*00201) are shed from the surface of these cells into the media. The mechanism of shedding of these proteins is, however, not known. Monoclonal antibodies W6/32, IL-A88, H1A, H6A, H11A, H58A, and PT-85A recognized surface MHC-I isoforms with varying affinity. We were able to develop a sandwich enzyme-linked immunosorbent assay (ELISA) using either H1A or IL-A88 antibody as the capture antibody and the W6/32 antibody for detection. We produced monoclonal antibodies against cattle NC1*00501 and NC3*00101 proteins. One monoclonal antibody generated against BoLA-NC3*00101 was highly specific. Unfortunately, due to failure to clone the NC3*00101- hybridoma, we no longer have an infinite source of this monoclonal antibody for NC3*00101. We eluted peptides from NC3*00101-transfected MHC-null K562 cells and identified peptides using liquid chromatography-mass spectrum (LC-MS) analysis. Analysis of peptide binding data using the SAS Proc mixed statistical program, suggested that the peptide EVTNQLVVL is a potential peptide ligand, which can be used to make tetramers for enumeration of antigen-specific leukocytes

Contribution of macrophages to fetomaternal immunological tolerance

The semi-allogeneic fetus develops in a uniquely immune tolerant environment within the uterus. For successful pregnancy, both the innate and adaptive immune systems must favor acceptance of the fetal allograft. Macrophages are the second most abundant immune cells after natural killer (NK) cells in the decidua. In coordination with decidual NK cells and dendritic cells, macrophages aid in implantation, vascular remodeling, placental development, immune tolerance to placental cells, and maintenance of tissue homeostasis at the maternal-fetal interface. Decidual macrophages show the classical activated (M1) and alternatively activated (M2) phenotypes under the influence of the local milieu of growth factors and cytokines, and appropriate temporal regulation of the M1/M2 switch is vital for successful pregnancy. Disturbances in the mechanisms that control the M1/M2 balance and associated functions during pregnancy can trigger a spectrum of pregnancy complications ranging from preeclampsia and fetal growth restriction to preterm delivery. This review addresses various mechanisms of tolerance, focusing on the basic biology of macrophages, their plasticity and polarization, and their protective roles at the immune-privileged maternal-fetal interface, including direct and indirect roles in promoting fetomaternal immune tolerance

A Non-Recoverable Hybridoma Limits the Production of Monoclonal Antibodies Against Bovine Trophoblast Non-Classical NC3*00101 Protein

The non-classical MHC class I (MHC-Ib) proteins are important modulators of immune system during pregnancy favoring survival of the fetus. Contrary to ubiquitously expressed classical MHC-I proteins, MHC-Ib proteins are oligomorphic, and expressed in specific cell/tissue types thus minimizing maternal immune-mediated rejection of fetal-allograft and a successful pregnancy. A unique characteristic of MHC-Ib glycoproteins is expression of surface and soluble isoforms due to alternative splicing phenomenon. Bovine fetal trophoblast cells, during the third trimester of pregnancy, express non-classical bovine leukocyte antigen class Ib (BoLA-Ib) antigens. BoLA-NC3*00101, is a non-classical class I allele from cattle AH11 haplotype and is expressed as cell surface isoform. However, lack of monoclonal antibody (mAb) hinders the development of specific assay to detect the soluble/secreted BoLA-I antigens released by fetal trophoblast cells. The objective of this study is to synthesize mAb specific to NC3*00101 molecule to develop an isotype-matched ELISA to assess BoLA-I protein(s). We demonstrated that majority of NC3*00101 hybridoma-supernatants were low-titer and showed inappreciable reactivity in ELISA and flow cytometry assays. We identified a clone of hybridoma (NC3-3*2) that secreted mAb specific to BoLA-NC3*00101 as demonstrated with flow cytometry. NC3-3*2 clone secreted supernatant that captured BoLA-NC3*00101 protein in ELISA. Unfortunately, despite several efforts we could not recover the cryopreserved hybridoma. Non-recoverability and instability, thus, limited production of NC3*00101-mAbs. This study provides the evidence that mice immunized with bovine class Ib proteins elicit a specific immunogenic response and warrants future studies into generation of stable hybridoma secreting antibodies against BoLA-Ib proteins using robust methods of fusion and cryopreservation of hybridomas

A non-recoverable hybridoma limits the production of monoclonal antibodies against bovine trophoblast non-classical <em>NC3*00101</em> protein

547-555The non-classical MHC class I (MHC-Ib) proteins are important modulators of immune system during pregnancy favoring survival of the fetus. Contrary to ubiquitously expressed classical MHC-I proteins, MHC-Ib proteins are oligomorphic, and expressed in specific cell/tissue types thus minimizing maternal immune-mediated rejection of fetal-allograft and a successful pregnancy. A unique characteristic of MHC-Ib glycoproteins is expression of surface and soluble isoforms due to alternative splicing phenomenon. Bovine fetal trophoblast cells, during the third trimester of pregnancy, express non-classical bovine leukocyte antigen class Ib (BoLA-Ib) antigens. BoLA-NC3*00101, is a non-classical class I allele from cattle AH11 haplotype and is expressed as cell surface isoform. However, lack of monoclonal antibody (mAb) hinders the development of specific assay to detect the soluble/secreted BoLA-I antigens released by fetal trophoblast cells. The objective of this study is to synthesize mAb specific to NC3*00101 molecule to develop an isotype-matched ELISA to assess BoLA-I protein(s). We demonstrated that majority of NC3*00101 hybridoma-supernatants were low-titer and showed inappreciable reactivity in ELISA and flow cytometry assays. We identified a clone of hybridoma (NC3-3*2) that secreted mAb specific to BoLA-NC3*00101 as demonstrated with flow cytometry. NC3-3*2 clone secreted supernatant that captured BoLA-NC3*00101 protein in ELISA. Unfortunately, despite several efforts we could not recover the cryopreserved hybridoma. Non-recoverability and instability, thus, limited production of NC3*00101-mAbs. This study provides the evidence that mice immunized with bovine class Ib proteins elicit a specific immunogenic response and warrants future studies into generation of stable hybridoma secreting antibodies against BoLA-Ib proteins using robust methods of fusion and cryopreservation of hybridomas

Therapeutic potential of deuterium-stabilized (R)-pioglitazone-PXL065-for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and β-oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C-ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to-date for ALD. Pioglitazone, an anti-diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and nongenomic (mitochondrial pyruvate carrier-MPC, long-chain acyl-CoA synthetase 4-ACSL4, inhibition). However, its use is limited by PPARγ-driven side effects (e.g. weight gain, edema). PXL065 is a clinical-stage deuterium-stabilized (R)-enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient-derived cells (both AMN and C-ALD) and glial cells from Abcd1-null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1-null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted

Beneficial Effects of the Direct AMP-Kinase Activator PXL770 in In Vitro and In Vivo Models of X-Linked Adrenoleukodystrophy

Background: X-linked adrenoleukodystrophy (ALD) is a severe orphan disease caused by mutations in the peroxisomal ABCD1 transporter gene, leading to toxic accumulation of Very Long-Chain Fatty Acids (VLCFA - in particular C26:0) resulting in inflammation, mitochondrial dysfunction and demyelination. AMP-activated protein kinase (AMPK) is downregulated in ALD, and its activation is implicated as a therapeutic target. PXL770 is the first direct allosteric AMPK activator with established clinical efficacy and tolerability. Methods: We investigated its effects in ALD patient-derived fibroblasts/lymphocytes and Abcd1 KO mouse glial cells. Readouts included VLCFA levels, mitochondrial function and mRNA levels of proinflammatory genes and compensatory transporters (ABCD2-3). Following PXL770 treatment in Abcd1 KO mice, we assessed VLCFA levels in tissues, sciatic nerve axonal morphology by electronic microscopy and locomotor function by open-field/balance-beam tests. Results: In patients\u27 cells and Abcd1 KO glial cells, PXL770 substantially decreased C26:0 levels (by ~90%), improved mitochondrial respiration, reduced expression of multiple inflammatory genes and induced expression of ABCD2-3 In Abcd1 KO mice, PXL770 treatment normalized VLCFA in plasma and significantly reduced elevated levels in brain (-25%) and spinal cord (-32%) vs. untreated (p\u3c0.001). Abnormal sciatic nerve axonal morphology was also improved along with amelioration of locomotor function. Conclusion: Direct AMPK activation exerts beneficial effects on several hallmarks of pathology in multiple ALD models in vitro and in vivo, supporting clinical development of PXL770 for this disease. Further studies would be needed to overcome limitations including small sample size for some parameters, lack of additional in vivo biomarkers and incomplete pharmacokinetic characterization. Significance Statement Adrenoleukodystrophy is a rare and debilitating condition with no approved therapies, caused by accumulation of very long-chain fatty acids. AMPK is downregulated in the disease and has been implicated as a potential therapeutic target. PXL770 is a novel clinical stage direct AMPK activator. In these studies, we used PXL770 to achieve preclinical validation of direct AMPK activation for this disease - based on correction of key biochemical and functional readouts in vitro and in vivo, thus supporting clinical development

The Czech Confederation as a Constitutional Basis for Union of Central Europe

Katedra právních dějinDepartment of Legal HistoryFaculty of LawPrávnická fakult

Supplemental Material, Supplementary_Material_TableS2 - An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis

<p>Supplemental Material, Supplementary_Material_TableS2 for An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis by Shane Lipskind, Jennifer S. Lindsey, Behzad Gerami-Naini, Jennifer L. Eaton, Daniel O’Connell, Adam Kiezun, Joshua W. K. Ho, Nicholas Ng, Parveen Parasar, Michelle Ng, Michael Nickerson, Utkan Demirci, Richard Maas, and Raymond M. Anchan in Reproductive Sciences</p

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. •Bovine non-classical MHC-I proteins can be either membrane associated or secreted.•BoLA-NC1*00501, -NC3*00101 and -NC4*00201 were membrane associated proteins.•The BoLA-NC1*00401 splice variant was expressed as a secreted protein.•BoLA-NC2*00102 remained intracellular and may require a specific peptide antigen