Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce.

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

miRNA-130a regulates C/EBP-ε expression during granulopoiesis.

CCAAT/enhancer binding protein-ε (C/EBP-ε) is considered a master transcription factor regulating terminal neutrophil maturation. It is essential for expression of secondary granule proteins, but it also regulates proliferation, cell cycle, and maturation during granulopoiesis. Cebpe(-/-) mice have incomplete granulocytic differentiation and increased sensitivity toward bacterial infections. The amount of C/EBP-ε messenger RNA (mRNA) increases with maturation from myeloblasts with peak level in myelocytes (MC)/metamyelocytes (MM), when the cells stop proliferating followed by a decline in more mature cells. In contrast, C/EBP-ε protein is virtually detectable only in the MC/MM population, indicating that expression in more immature cells could be inhibited by microRNAs (miRNAs). We found that miRNA-130a (miR-130a) regulates C/EBP-ε protein expression in both murine and human granulocytic precursors. Overexpression of miR-130a in a murine cell line downregulated C/EBP-ε protein and lactoferrin (Ltf), cathelicidin antimicrobial protein (Camp), and lipocalin-2 (Lcn2) mRNA expression giving rise to cells with a more immature phenotype, as seen in the Cebpe(-/-) mouse. Introduction of a C/EBP-ε mRNA without target site for miR-130a restored both C/EBP-ε production, expression of Camp and Lcn2, and resulted in the cells having a more mature phenotype. We conclude that miR-130a is important for the regulation of the timed expression of C/EBP-ε during granulopoiesis.

MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-ß1 stimulation in granulocytic precursors.

Smad4 is important in the TGF-ß pathway and required for transcriptional activation and inhibition of cell growth after TGF-ß1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3'-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-ß1-induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia-derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-ß1-mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-ß pathway.

FVIII activity following FVIII protein infusion or FVIII gene transfer predicts the bleeding risk in hemophilia A rats.

BACKGROUND: Prophylactic replacement therapy in hemophilia A (HA) patients does not adequately prevent bleeds and arthropathic complications. A more refined understanding of the relationship between coagulation factor VIII (FVIII) levels and bleeding risk during protein prophylaxis, or with gene therapy, is needed to improve patient care. OBJECTIVES: Investigate this relationship in the HA rat, a model exhibiting spontaneous bleeds and development of arthropathy similar to HA patients. METHODS: Human B domain-deleted FVIII was delivered to HA rats via adeno-associated virus (AAV)-mediated gene transfer or multiple intravenous protein injections. RESULTS AND CONCLUSIONS: After 12 weeks of observation, both approaches significantly reduced bleeds per animal and increased the proportion of bleed-free animals compared with controls (43% vs 0%, respectively [AAV]; 75% vs 8%, respectively [injection]). Both approaches resulted in an anti-FVIII inhibitory response in 20% to 37% of treated animals, similar to HA patients. Inhibitory antibodies were refractory to clinical improvement (reduction of bleeds) only in the AAV-based prophylaxis. An integrated model-based analysis of data on FVIII exposure and bleeding events was performed. This predicted the bleeding risk at any given circulating FVIII activity. Specifically, 4.8 or 10 IU/dL FVIII (0.048 and 0.1 IU/mL, respectively) were predicted to reduce bleeding risk by 90% or 95%, respectively, compared with untreated controls. Our data establish the utility of the HA rat model in FVIII prophylaxis studies and describe how FVIII activity affects bleeding risk in this setting. These enable further studies on FVIII prophylaxis focusing on disease complications for an optimized treatment of HA patients.

MicroRNA profiling in human neutrophils during bone marrow granulopoiesis and in vivo exudation.

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.

No effect of NGAL/lipocalin-2 on aggressiveness of cancer in the MMTV-PyMT/FVB/N mouse model for breast cancer.

NGAL/lipocalin-2 is a siderophore-binding protein that is highly expressed in several cancers. It is suggested to confer a proliferative advantage to cancer cells. Its expression has been correlated with aggressiveness of breast cancer as determined both in patients and in mouse breast cancer models. This was recently confirmed in two mouse models of spontaneous breast cancer in wild-type and lipocalin-2-deficient mice. We used a similar strategy using a different mouse strain. Lipocalin-2-deficient mice and mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) mice were crossed into the same FVB/N background. All mice developed tumors by week 8. The mice were sacrificed on week 13 and tissue was processed for biochemical and histological analysis. The total tumor volume and number of metastases were quantitated in 26 lipocalin-2-deficient mice and 34 wild-type controls. Lipocalin-2 expression in tumors of MMTV-PyMT-positive and wild-type mice was assessed by quantitative real-time PCR and by immunohistochemistry. The expression of the lipocalin-2 receptors 24p3R and megalin and of Mmp-9, transferrin receptor, and Bdh2 (a producer of a mammalian siderophore) were quantitated by real-time PCR. No significant difference was observed between wild-type and lipocalin-2-deficient mice. Lipocalin-2 was highly expressed in tumors from wild-type mice, but the expression did not correlate with tumor size. No effect of lipocalin-2 was observed with respect to time to tumor appearance, total tumor volume, or to the number of metastases. Histology and gelatinolytic activity of the mammary tumors did not differ between wild-type and lipocalin-2-deficient mice. We conclude that NGAL/lipocalin-2 does not invariably affect the aggressiveness of breast cancers as assessed in mouse models, thus questioning the role of lipocalin-2 in cancer development.

Intrinsic laryngeal muscles are spared from degeneration in the dy3K/dy3K mouse model of congenital muscular dystrophy type 1A.

Deficiency of laminin alpha2 chain leads to a severe form of congenital muscular dystrophy (MDC1A). Here, we analyzed whether the intrinsic laryngeal muscles (ILM) are spared in the dy(3K)/dy(3K) mouse model of complete laminin alpha2 chain absence. No muscle degeneration was evident; expression of various laminin chains was similar to that of limb muscles, and sustained integrin alpha7B expression was noted in laminin alpha2 chain-deficient ILM. We conclude that ILM are spared in MDC1A.

Cib2 binds integrin alpha7Bbeta1D and is reduced in laminin alpha2 chain-deficient muscular dystrophy.

Mutations in the gene encoding laminin alpha2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin alpha2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin alpha7beta1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin alpha2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin alphaIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin alpha7beta1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin alpha7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin alpha7Bbeta1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin alpha7Bbeta1D signaling in skeletal muscle.

Laminin {alpha}1 chain corrects male infertility caused by absence of laminin {alpha}2 chain.

Laminins are important for basement membrane structure and function. The laminin alpha2 chain is a major component of muscle basement membranes, and mutations in the laminin alpha2 gene lead to congenital muscular dystrophy in humans and mice. Although the laminin alpha2 chain is prominently expressed in testicular basement membranes, its role in testis has remained unclear. Here, we show that laminin alpha1, alpha2, beta1, beta2, gamma 1, and gamma 3 chains are the major laminin chains in basement membranes of seminiferous tubules. In laminin alpha2 chain-deficient dy(3 K)/dy(3 ASK) mice, lack of laminin alpha2 chain led to concurrent reduction of laminin gamma 3 chain and abnormal testicular basement membranes. Seminiferous tubules of laminin alpha2 chain-deficient dy(3 K)/dy(3 K) mice displayed a defect in the timing of lumen formation, resulting in production of fewer spermatides. We also demonstrate that overexpression of laminin alpha1 chain in testis of dy(3 K)/dy(3 K) mice compensated for laminin alpha2 chain deficiency and significantly reversed the appearance of the histopathological features. We thus provide genetic data that laminin alpha chains are essential for normal testicular function in vivo.