Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics.

BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

BACKGROUND AND OBJECTIVES: Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis. METHODS: In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs. RESULTS: Using an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. DISCUSSION: Therefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell-mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome.

Rapid Depletion of Intratumoral Regulatory T Cells Induces Synchronized CD8 T- and NK-cell Activation and IFNγ-Dependent Tumor Vessel Regression.

Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab')2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies. SIGNIFICANCE: Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/3092/F1.large.jpg.

Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis.

Non-infectious uveitis, a common cause of blindness in man, is often mediated by autoimmunity, a process in which cytokines play major roles. The biosynthesis and secretion of pro-inflammatory cytokines are regulated in part by tristetraprolin (TTP), an endogenous anti-inflammatory protein that acts by binding directly to specific sequence motifs in the 3'-untranslated regions of target mRNAs, promoting their turnover, and inhibiting synthesis of their encoded proteins. We recently developed a TTP-overexpressing mouse (TTPΔARE) by deleting an AU-rich element (ARE) instability motif from the TTP mRNA, resulting in increased accumulation of TTP mRNA and protein throughout the animal. Here, we show that homozygous TTPΔARE mice are resistant to the induction of experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP), an established model for human autoimmune (noninfectious) uveitis. Lymphocytes from TTPΔARE mice produced lower levels of the pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and TNFα than wild type (WT) mice. TTPΔARE mice also produced lower titers of antibodies against the uveitogenic protein. In contrast, TTPΔARE mice produced higher levels of the anti-inflammatory cytokine IL-10, and had higher frequencies of regulatory T-cells, which, moreover, displayed a moderately higher per-cell regulatory ability. Heterozygous mice developed EAU and associated immunological responses at levels intermediate between homozygous TTPΔARE mice and WT controls. TTPΔARE mice were able, however, to develop EAU following adoptive transfer of activated WT T-cells specific to IRBP peptide 651-670, and naïve T-cells from TTPΔARE mice could be activated by antibodies to CD3/CD28. Importantly, TTPΔARE antigen presenting cells were significantly less efficient compared to WT in priming naïve T cells, suggesting that this feature plays a major role in the dampened immune responses of the TTPΔARE mice. Our observations demonstrate that elevated systemic levels of TTP can inhibit the pathogenic processes involved in EAU, and suggest the possible use of TTP-based treatments in humans with uveitis and other autoimmune conditions.

Immuno-PET Imaging of the Programmed Cell Death-1 Ligand (PD-L1) Using a Zirconium-89 Labeled Therapeutic Antibody, Avelumab.

OBJECTIVE: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. METHODS: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). RESULTS: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. CONCLUSIONS: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.

Pseudovirus rVSVΔG-ZEBOV-GP Infects Neurons in Retina and CNS, Causing Apoptosis and Neurodegeneration in Neonatal Mice.

Zaire Ebola virus (ZEBOV) survivors experience visual and CNS sequelae that suggests the ZEBOV glycoprotein can mediate neurotropism. Replication-competent rVSVΔG-ZEBOV-GP vaccine candidate is generally well tolerated; however, its potential neurotropism requires careful study. Here, we show that a single inoculation of rVSVΔG-ZEBOV-GP virus in neonatal C57BL/6 mice results in transient viremia, neurological symptoms, high viral titers in eyes and brains, and death. rVSVΔG-ZEBOV-GP infects the inner layers of the retina, causing severe retinitis. In the cerebellum, rVSVΔG-ZEBOV-GP infects neurons in the granular and Purkinje layers, resulting in progressive foci of apoptosis and neurodegeneration. The susceptibility to infection is not due to impaired type I IFN responses, although MDA5-/-, IFNß-/-, and IFNAR1-/- mice have accelerated mortality. However, boosting interferon levels by co-administering poly(I:C) reduces viral titers in CNS and improves survival. Although these data should not be directly extrapolated to humans, they challenge the hypothesis that VSV-based vaccines are non-neurotropic.

The Distribution Volume of 18F-Albumin as a Potential Biomarker of Antiangiogenic Treatment Efficacy.

Objective: 18F-albumin, a vascular imaging agent, may have potential to assess tumor responses to anti-angiogenic therapies. In these studies tumor distribution volume of 18F-albumin were first determined in various human tumor xenografts from biodistribtuion measurments and then one of the tumor type was used to evaluate changes in 18F-albumin uptake in anti-angiognic tumor model. Method: 18F-albumin was synthesized via conjugation of 6-[18F]fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [18F]F-Py-TFP, with rat albumin. From the biodistribution of 18F-albumin in various human tumor xenografts tumor distribution volumes (DVs; tumor%ID/g:blood%ID/g) were first determined at various time points. Then, the ability of 18F-albumin to detect tumor angiogenic inhibition in one of these tumor types (U87MG) following treatment with sunitinib was evaluated by position emission tomography (PET) imaging at 0, 7, 14, and 21 days post treatment. Caliper measurements of tumor dimensions were also made at these same times. At Day 21, following imaging, biodistributions, autoradiography of tumor tissues and tumor blood vessel counts (CD31 IHC) were performed. Results: 18F-albumin retention in various tumors steadily increased over time with U87MG tumor exhibiting the highest uptake (DV) at all times. Significant decreases in 18F-albumin DVs were observed one week post-treatement (-39%) vs. controls whereas tumor caliper volumes were not significantly decreased until days 14 and 21. At day 21 the significant decrease in DVs in the treatment group (-44%) paralleled biodistribution DV measurements and was consistent with autoradiography and CD31 IHC findings. Conclusion: These data suggest that 18F-albumin DVs obtained by imaging may serve as an early biomarker of the effectiveness of anti-angiogenic therapy and thus aid in patient management and treatment planning.

TMP778, a selective inhibitor of RORγt, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations.

Experimental autoimmune uveitis (EAU), an animal model for severe intraocular inflammatory eye diseases, is mediated by both Th1 and Th17 cells. Here, we examined the capacity of TMP778, a selective inhibitor of RORγt, to inhibit the development of EAU, as well as the related immune responses. EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). Treatment with TMP778 significantly inhibited the development of EAU, determined by histological examination. In addition, the treatment suppressed the cellular immune response to IRBP, determined by reduced production of IL-17 and IFN-γ, as well as lower percentages of lymphocytes expressing these cytokines, as compared to vehicle-treated controls. The inhibition of IFN-γ expression by TMP778 is unexpected in view of this compound being a selective inhibitor of RORγt. The observation was further confirmed by the finding of reduced expression of the T-bet (Tbx21) gene, the transcription factor for IFN-γ, by cells of TMP778-treated mice. Thus, these data demonstrate the capacity of TMP778 to inhibit pathogenic autoimmunity in the eye and shed new light on its mode of action in vivo.

ZIKA virus infection causes persistent chorioretinal lesions.

Zika-infected patients can have eye involvement ranging from mild conjunctivitis to severe chorioretinal lesions, however the possible long-term sequelae of infection and timeline to recovery remain unknown. Here we describe the partial recovery of chorioretinal lesions in an immunocompetent patient diagnosed with bilateral posterior uveitis associated with Zika infection and show that some lesions resolved with focal atrophy evident as pigmentary changes on funduscopy. To better understand the progression of the lesions and correlate the changes in fundus imaging with local viral load, immune responses, and retinal damage, we developed a symptomatic mouse model of ocular Zika virus infection. Imaging of the fundus revealed multiple hypopigmentary patches indicative of chorioretinal degeneration as well as thinning of the retina that mirror the lesions in patients. Microscopically, the virus primarily infected the optic nerve, retinal ganglion cells, and inner nuclear layer cells, showing thinning of the outer plexiform layer. During acute infection, the eyes showed retinal layer disorganization, retinitis, vitritis, and focal choroiditis, with mild cellular infiltration and increased expression of tumor necrosis factor, interferon-γ, granzyme B, and perforin. Focal areas of gliosis and retinal degeneration persisted 60 dpi. The model recapitulates features of ZIKA infections in patients and should help elucidate the mechanisms underlying the damage to the eyes and aid in the development of effective therapeutics.

Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy.

Current immunotherapies for cancer seek to modulate the balance among different immune cell populations, thereby promoting antitumor immune responses. However, because these are systemic therapies, they often cause treatment-limiting autoimmune adverse effects. It would be ideal to manipulate the balance between suppressor and effector cells within the tumor without disturbing homeostasis elsewhere in the body. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are well-known immunosuppressor cells that play a key role in tumor immunoevasion and have been the target of systemic immunotherapies. We used CD25-targeted near-infrared photoimmunotherapy (NIR-PIT) to selectively deplete Tregs, thus activating CD8 T and natural killer cells and restoring local antitumor immunity. This not only resulted in regression of the treated tumor but also induced responses in separate untreated tumors of the same cell line derivation. We conclude that CD25-targeted NIR-PIT causes spatially selective depletion of Tregs, thereby providing an alternative approach to cancer immunotherapy.

Automated synthesis of [(18)F](2S,4R)-4-fluoroglutamine on a GE TRACERlab™ FX-N Pro module.

Glutamine (Gln) and its analogues may serve as imaging agents for tumor diagnosis using positron emission tomography (PET), especially for tumors with negative [(18)F]FDG scan. We report the first automated synthesis of [(18)F](2S,4R)-4-fluoroglutamine ([(18)F]FGln) on a GE TRACERlab™ FX-N Pro module. [(18)F]FGln was obtained in 80±3min with a radiochemical yield of 21±3% (n=5, uncorrected). The radiochemical purity was >98%, and optical purity 90±5%. The synthesis is highly reproducible with good chemical purity, radiochemical yield, and is suitable for translation to cGMP production.

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or Cy 5** (AH-112543) Labeled Peptide.

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIs). A peptide targeting Met labeled with [(99m)Tc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [(99m)Tc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using single-photon emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [(99m)Tc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [(99m)Tc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [(99m)Tc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [(99m)Tc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

STAQ: A route toward low power, multicolor nanoscopy.

Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (∼50 nm) imaging. Moreover, the TAQ partner excited by the "donut" beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist's palette.

Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.

We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent.

A complex role of herpes viruses in the disease process of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). Neither the antigenic target(s) nor the cell population(s) responsible for CNS tissue destruction in MS have been fully defined. The objective of this study was to simultaneously determine the antigen (Ag)-specificity and phenotype of un-manipulated intrathecal CD4+ and CD8+ T cells of patients with relapsing-remitting and progressive MS compared to subjects with other inflammatory neurological diseases. We applied a novel Ag-recognition assay based on co-cultures of freshly obtained cerebrospinal fluid T cells and autologous dendritic cells pre-loaded with complex candidate Ag's. We observed comparably low T cell responses to complex auto-Ag's including human myelin, brain homogenate, and cell lysates of apoptotically modified oligodendroglial and neuronal cells in all cohorts and both compartments. Conversely, we detected a strong intrathecal enrichment of Epstein-Barr virus- and human herpes virus 6-specific (but not cytomegalovirus-specific) reactivities of the Th1-phenotype throughout all patients. Qualitatively, the intrathecal enrichment of herpes virus reactivities was more pronounced in MS patients. This enrichment was completely reversed by long-term treatment with the IL-2 modulating antibody daclizumab, which strongly inhibits MS disease activity. Finally, we observed a striking discrepancy between diminished intrathecal T cell proliferation and enhanced cytokine production of herpes virus-specific T cells among progressive MS patients, consistent with the phenotype of terminally differentiated cells. The data suggest that intrathecal administration of novel therapeutic agents targeting immune cells outside of the proliferation cycle may be necessary to effectively eliminate intrathecal inflammation in progressive MS.

Immuno-PET imaging of tumor endothelial marker 8 (TEM8).

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Simultaneous ECG-gated PET imaging of multiple mice.

INTRODUCTION: We describe and illustrate a method for creating ECG-gated PET images of the heart for each of several mice imaged at the same time. The method is intended to increase "throughput" in PET research studies of cardiac dynamics or to obtain information derived from such studies, e.g. tracer concentration in end-diastolic left ventricular blood. METHODS: An imaging bed with provisions for warming, anesthetic delivery, etc., was fabricated by 3D printing to allow simultaneous PET imaging of two side-by-side mice. After electrode attachment, tracer injection and placement of the animals in the scanner field of view, ECG signals from each animal were continuously analyzed and independent trigger markers generated whenever an R-wave was detected in each signal. PET image data were acquired in "list" mode and these trigger markers were inserted into this list along with the image data. Since each mouse is in a different spatial location in the FOV, sorting of these data using trigger markers first from one animal and then the other yields two independent and correctly formed ECG-gated image sequences that reflect the dynamical properties of the heart during an "average" cardiac cycle. RESULTS: The described method yields two independent ECG-gated image sequences that exhibit the expected properties in each animal, e.g. variation of the ventricular cavity volumes from maximum to minimum and back during the cardiac cycle in the processed animal with little or no variation in these volumes during the cardiac cycle in the unprocessed animal. CONCLUSION: ECG-gated image sequences for each of several animals can be created from a single list mode data collection using the described method. In principle, this method can be extended to more than two mice (or other animals) and to other forms of physiological gating, e.g. respiratory gating, when several subjects are imaged at the same time.