Therapeutic effect of IVIG on inflammatory arthritis in mice is dependent on the Fc portion and independent of sialylation or basophils.

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN-expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1-2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab')2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

Targeting the relaxin hormonal pathway in prostate cancer.

Targeting the androgen signalling pathway has long been the hallmark of anti-hormonal therapy for prostate cancer. However, development of androgen-independent prostate cancer is an inevitable outcome to therapies targeting this pathway, in part, owing to the shift from cancer dependence on androgen signalling for growth in favor of augmentation of other cellular pathways that provide proliferation-, survival- and angiogenesis-promoting signals. This review focuses on the role of the hormone relaxin in the development and progression of prostate cancer, prior to and after the onset of androgen independence, as well as its role in cancers of other reproductive tissues. As the body of literature expands, examining relaxin expression in cancerous tissues and its role in a growing number of in vitro and in vivo cancer models, our understanding of the important involvement of this hormone in cancer biology is becoming clearer. Specifically, the pleiotropic functions of relaxin affecting cell growth, angiogenesis, blood flow, cell migration and extracellular matrix remodeling are examined in the context of cancer progression. The interactions and intercepts of the intracellular signalling pathways of relaxin with the androgen pathway are explored in the context of progression of castration-resistant and androgen-independent prostate cancers. We provide an overview of current anti-hormonal therapeutic treatment options for prostate cancer and delve into therapeutic approaches and development of agents aimed at specifically antagonizing relaxin signalling to curb tumor growth. We also discuss the rationale and challenges utilizing such agents as novel anti-hormonals in the clinic, and their potential to supplement current therapeutic modalities.

Cytokine profiles in mouse models of experimental immune thrombocytopenia reveal a lack of inflammation and differences in response to intravenous immunoglobulin depending on the mouse strain.

BACKGROUND: Mouse models of human immune thrombocytopenia (ITP) have been used for years to investigate the mechanism of intravenous immunoglobulin (IVIG) to ameliorate ITP; however, how closely these experimental mouse models mirror the human autoimmune inflammatory disease is unclear. The aim of this study was to assess the cytokine profiles in experimental ITP with and without IVIG treatment. STUDY DESIGN AND METHODS: We examined the production of 23 cytokines that included pro- and anti-inflammatory cytokines, in two different mouse strain models of ITP, BALB/c and C57BL/6J, with and without IVIG treatment. RESULTS: In contrast to human ITP, in mouse models of ITP generated by passive transfer of an alloantibody we find no evidence of inflammatory disease even when ITP is robust, suggesting that while these models capture the innate response phase of the disease, they may not be capturing the adaptive mechanisms of autoimmune initiation of the disorder. Regardless of the mouse strain examined, interleukin (IL)-1α, -2, -6, -17, and -23; granulocyte-macrophage-colony-stimulating factor (GM-CSF); monocyte chemoattractant protein (MCP)-1; macrophage inflammatory protein (MIP)-1ß; RANTES; tumor necrosis factor (TNF)-α; and interferon-γ remain at negligible levels after ITP induction. IVIG treatment in the absence of ITP induced significant levels of IL-4, -10, -11, -17, and -23; GM-CSF; MCP-1; and TNF-α in BALB/c mice, but only IL-11 was elevated in C57BL/6J mice. In response to IVIG treatment of ITP, both strains produced IL-4, -10, -11, and -23; GM-CSF; MCP-1; MIP-1ß; and TNF-α; however, only BALB/c exhibited increased MCP-3 and RANTES. IL-11 levels were the highest of any cytokine after IVIG administration, given either alone or as treatment of ITP. CONCLUSIONS: We conclude that mouse models for human ITP do not capture the full range of autoimmune inflammatory mechanisms of this disease. Furthermore, cytokine profiles differ in response to IVIG depending on the mouse strain used.

Structure-activity relationships of pyrazole derivatives as potential therapeutics for immune thrombocytopenias.

Idiopathic or immune thrombocytopenia (ITP) is a serious clinical disorder involving the destruction of platelets by macrophages. Small molecule therapeutics are highly sought after to ease the burden on current therapies derived from human sources. Earlier, we discovered that dimers of five-membered heterocycles exhibited potential to inhibit phagocytosis of human RBCs by macrophages. Here, we reveal a structure-activity relationship of the bis-pyrazole class of molecules with -C-C-, -C-N- and -C-O- linkers, and their evaluation as inhibitors of phagocytosis of antibody-opsonized human RBCs as potential therapeutics for ITP. We have uncovered three potential candidates, 37, 47 and 50, all carrying a different linker connecting the two pyrazole moieties. Among these compounds, hydroxypyrazole derivative 50 is the most potent compound with an IC50 of 14 ± 9 µM for inhibiting the phagocytosis of antibody-opsonized human RBCs by macrophages. None of the compounds exhibited significant potential to induce apoptosis in peripheral blood mononuclear cells (PBMCs). Current study has revealed specific functional features, such as up to 2-atom spacer arm and alkyl substitution at one of the N(1) positions of the bivalent pyrazole core to be important for the inhibitory activity.

Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells.

Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strategy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the biological evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency in the increasing order of 10 ~/= 11~/= 16 < 19 < 20. A novel scaffold, 20 with an IC(50) of 100 nM inhibiting opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirmation of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying ligands targeting ITP and other similar disorders.

Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase.

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Engineered Thymidine-Active Deoxycytidine Kinase for Bystander Killing of Malignant Cells.

Suicide transgenes encode proteins that are either capable of activating specific prodrugs into cytotoxic antimetabolites that can trigger cancer cell apoptosis or are capable of directly inducing apoptosis. Suicide gene therapy of cancer (SGTC) involves the targeted or localized delivery of suicide transgene sequences into tumor cells by means of various gene delivery vehicles. SGTC that operates via the potentiation of small-molecule pharmacologic agents can elicit the elimination of cancer cells within a tumor beyond only those cells successfully transduced. Such "bystander effects ", typically mediated by the spread of activated cytotoxic antimetabolites from the transduced cells expressing the suicide transgene to adjacent cells in the tumor, can lead to a significant reduction of the tumor mass without the requirement of transduction of a high percentage of cells within the tumor. The spread of activated cytotoxic molecules to adjacent cells is mediated primarily by diffusion and normally involves gap junctional intercellular communications (GJIC). We have developed a novel SGTC system based on viral vector-mediated delivery of an engineered variant of human deoxycytidine kinase (dCK), which is capable of phosphorylating uridine- and thymidine-based nucleoside analogues that are not substrates for wild-type dCK, such as bromovinyl deoxyuridine (BVdU) and L-deoxythymidine (LdT). Since our dCK-based SGTC system is capable of mediating strong bystander cell killing, it holds promise for clinical translation. In this chapter, we detail the key procedures for the preparation of recombinant lentivectors for the delivery of engineered dCK, transduction of tumor cells, and evaluation of bystander cell killing effects in vitro and in vivo.

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth.

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (deltaH2) impaired its biological function as measured by cAMP signaling. In a competition assay, deltaH2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, deltaH2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or deltaH2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing deltaH2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that deltaH2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express deltaH2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.

Mouse Models for Immune-Mediated Platelet Destruction or Immune Thrombocytopenia (ITP).

Immune thrombocytopenia (ITP) is a debilitating, life-threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have been developed that contributed to the basic understanding of the underlying mechanisms of ITP onset, progression, and maintenance. Rodent models that develop ITP spontaneously, or by passive transfer of an antiplatelet sera or antibody, play an instrumental role in the investigation of ITP mechanisms responsible for the breakdown of tolerance in human ITP, in studies of the immunopathology underlying the progression of platelet destruction, and in elucidation of the mechanisms of therapeutic amelioration of ITP by existing and new therapeutic modalities. This unit captures the protocols for the implementation and readout of passive antibody transfer mouse models of ITP, established by the infusion of a commercially-available monoclonal rat anti-mouse CD41 platelet antibody.

Small molecule phagocytosis inhibitors for immune cytopenias.

Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis.

Engineering Hematopoietic Cells for Cancer Immunotherapy: Strategies to Address Safety and Toxicity Concerns.

Advances in cancer immunotherapies utilizing engineered hematopoietic cells have recently generated significant clinical successes. Of great promise are immunotherapies based on chimeric antigen receptor-engineered T (CAR-T) cells that are targeted toward malignant cells expressing defined tumor-associated antigens. CAR-T cells harness the effector function of the adaptive arm of the immune system and redirect it against cancer cells, overcoming the major challenges of immunotherapy, such as breaking tolerance to self-antigens and beating cancer immune system-evasion mechanisms. In early clinical trials, CAR-T cell-based therapies achieved complete and durable responses in a significant proportion of patients. Despite clinical successes and given the side effect profiles of immunotherapies based on engineered cells, potential concerns with the safety and toxicity of various therapeutic modalities remain. We discuss the concerns associated with the safety and stability of the gene delivery vehicles for cell engineering and with toxicities due to off-target and on-target, off-tumor effector functions of the engineered cells. We then overview the various strategies aimed at improving the safety of and resolving toxicities associated with cell-based immunotherapies. Integrating failsafe switches based on different suicide gene therapy systems into engineered cells engenders promising strategies toward ensuring the safety of cancer immunotherapies in the clinic.

c-SRC protein tyrosine kinase regulates early HIV-1 infection post-entry.

OBJECTIVE: We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is through the non-receptor tyrosine kinase pp60 (c-SRC) and its binding partner, protein tyrosine kinase 2 beta (PTK2B). DESIGN: CD4 T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. We used SRC-family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B, then monitored effects on the early HIV-1 lifecycle. METHODS: Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce c-SRC or PTK2B protein expression. Activated CD4 T-lymphocytes were infected with recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments examined early infection by monitoring: luciferase activity, expression of host surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts, integrated HIV-1, and two-long terminal repeat (2-LTR) circles. RESULTS: All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4, and CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (eight-fold) while also decreasing viral integration (four-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown produced similar results. CONCLUSIONS: Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection, participating in multiple stages of infection post-entry: Reduced proviral integration with increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral therapy. Decreasing c-SRC expression and/or activity provides a new target for antiviral intervention and the potential for repurposing existing FDA-approved kinase inhibitors.

Extracellular histones identified in crocodile blood inhibit in-vitro HIV-1 infection.

OBJECTIVE: It has been reported that crocodile blood contains potent antibacterial and antiviral properties. However, its effects on HIV-1 infection remain unknown. DESIGN: We obtained blood from saltwater crocodiles to examine whether serum or plasma could inhibit HIV-1 infection. We purified plasma fractions then used liquid chromatography-mass spectrometry to identify the inhibitory protein factor(s). We then analyzed the ability of recombinant proteins to recapitulate HIV-1 inhibition and determine their mechanism of action. METHODS: Crocodylus porosus plasma was tested for inhibition of Jurkat T-cell HIV-1 infection. Inhibitor(s) were purified by reverse-phase chromatography then identified by protein liquid chromatography-mass spectrometry. Anti-HIV-1 activity of purified plasma or recombinant proteins were measured by p24 enzyme-linked immunosorbent assay and luciferase readouts, and mechanism of action was determined by measuring HIV-1 RNA, cDNA and transcription (using 1G5 cells). RESULTS: Crocodile plasma contains potent inhibitors of HIV-1IIIB infection, which were identified as histones. Recombinant human histones H1 and H2A significantly reduced HIV-1JR-FL infection (IC50 of 0.79 and 0.45 µmol/l, respectively), whereas H4 enhanced JR-FL luciferase activity. The inhibitory effects of crocodile plasma, recombinant H1 or recombinant H2A on HIV-1 infection were during or post-viral transcription. CONCLUSION: Circulating histones in crocodile blood, possibly released by neutrophil extracellular traps, are significant inhibitors of HIV-1 infection in-vitro. Extracellular recombinant histones have different effects on HIV-1 transcription and protein expression and are downregulated in HIV-1 patients. Circulating histones may be a novel resistance factor during HIV-1 infection, and peptide versions should be explored as future HIV-1 therapeutics that modulate viral transcription.

Mouse models of autoimmune diseases: immune thrombocytopenia.

Immune thrombocytopenia or ITP is a debilitating and life-threatening disorder affecting more than 4 in every 10, 000 adults annually. Following a basic understanding of the immunopathology underlying ITP, namely that production of anti-platelet antibodies results in accelerated platelet clearance and thrombocytopenia, animal models of ITP were quickly developed. Rodent models that develop ITP spontaneously or by passive transfer of anti-platelet sera or antibodies have become instrumental in investigating the mechanisms responsible for the breakdown of tolerance in human ITP, understanding the immunopathology that underlies the progression of platelet destruction, elucidating the mechanism(s) of therapeutic amelioration of the ITP, and driving the development of new therapeutic modalities. This review aims to capture the development history and methodology of currently available ITP disease models, and review their advantages and limitations in the study of various aspects of ITP. We also review how closely the various ITP models reflect the pathobiology of human ITP and their usefulness in advancing the development of new therapeutics, which are particularly needed to address the unmet need of patients who are refractory to the currently available repertoire of interventions.

Evaluation of Bystander Cell Killing Effects in Suicide Gene Therapy of Cancer: Engineered Thymidylate Kinase (TMPK)/AZT Enzyme-Prodrug Axis.

Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant cells, where they can also mediate destruction. This phenomenon, termed "bystander killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander cell killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target cells, and various approaches for the evaluation of bystander cell killing effects in SGCT in both in vitro and in vivo models.

Relaxin receptor antagonist AT-001 synergizes with docetaxel in androgen-independent prostate xenografts.

Androgen hormones and the androgen receptor (AR) pathway are the main targets of anti-hormonal therapies for prostate cancer. However, resistance inevitably develops to treatments aimed at the AR pathway resulting in androgen-independent or hormone-refractory prostate cancer (HRPC). Therefore, there is a significant unmet need for new, non-androgen anti-hormonal strategies for the management of prostate cancer. We demonstrate that a relaxin hormone receptor antagonist, AT-001, an analog of human H2 relaxin, represents a first-in-class anti-hormonal candidate treatment designed to significantly curtail the growth of androgen-independent human prostate tumor xenografts. Chemically synthesized AT-001, administered subcutaneously, suppressed PC3 xenograft growth by up to 60%. AT-001 also synergized with docetaxel, standard first-line chemotherapy for HRPC, to suppress tumor growth by more than 98% in PC3 xenografts via a mechanism involving the downregulation of hypoxia-inducible factor 1 alpha and the hypoxia-induced response. Our data support developing AT-001 for clinical use as an anti-relaxin hormonal therapy for advanced prostate cancer.

The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

We previously described a novel suicide (or 'cell fate control') gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

Autologous transplantation of lentivector/acid ceramidase-transduced hematopoietic cells in nonhuman primates.

Farber disease is a rare lysosomal storage disorder (LSD) that manifests due to acid ceramidase (AC) deficiencies and ceramide accumulation. We present a preclinical gene therapy study for Farber disease employing a lentiviral vector (LV-huAC/huCD25) in three enzymatically normal nonhuman primates. Autologous, mobilized peripheral blood (PB) cells were transduced and infused into fully myelo-ablated recipients with tracking for at least 1 year. Outcomes were assessed by measuring the AC specific activity, ceramide levels, vector persistence/integration, and safety parameters. We observed no hematological, biochemical, radiological, or pathological abnormalities. Hematological recovery occurred by approximately 3 weeks. Vector persistence was observed in PB and bone marrow (BM) cells by qualitative and quantitative PCR. We did not observe any clonal proliferation of PB and BM cells. Importantly, AC-specific activity was detected above normal levels in PB and BM cells analyzed post-transplantation and in spleens and livers at the endpoint of the study. Decreases of ceramide in PB cells as well as in spleen and liver tissues were seen. We expect that this study will provide a roadmap for implementation of clinical gene therapy protocols targeting hematopoietic cells for Farber disease and other LSDs.

A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs.

UNLABELLED: In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET. METHODS: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs. RESULTS: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine). CONCLUSION: The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues.

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.