SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging.

BACKGROUND: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. METHODS: Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans. RESULTS: No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC. CONCLUSION: We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy. RELEVANCE STATEMENT: Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness. KEY POINTS: • Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Magnetic Particle Imaging Is a Sensitive In Vivo Imaging Modality for the Detection of Dendritic Cell Migration.

PURPOSE: The purpose of this study was to evaluate magnetic particle imaging (MPI) as a method for the in vivo tracking of dendritic cells (DC). DC are used in cancer immunotherapy and must migrate from the site of implantation to lymph nodes to be effective. The magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With current protocols, less than 10% of DC are expected to reach target nodes. Therefore, imaging techniques for studying DC migration must be sensitive and quantitative. Here, we describe the first study using MPI to detect and track DC injected into the footpads of C57BL/6 mice migrating to the popliteal lymph nodes (pLNs). PROCEDURES: DC were labelled with Synomag-D™ and injected into each hind footpad of C57BL/6 mice (n = 6). In vivo MPI was conducted immediately and repeated 48 h later. The MPI signal was measured from images and related to the signal from a known number of cells to calculate iron content. DC numbers were estimated by dividing iron content in the image by the iron per cell measured from a separate cell sample. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI, histology, and fluorescence microscopy. RESULTS: Day 2 imaging showed a decrease in MPI signal in the footpads and an increase in signal at the pLNs, indicating DC migration. MPI signal was detected in the left pLN in four of the six mice and two of the six mice showed MPI signal in the right pLN. Ex vivo imaging detected signal in 11/12 nodes. We report a sensitivity of approximately 4000 cells (0.015 µg Fe) in vivo and 2000 cells (0.007 µg Fe) ex vivo. CONCLUSIONS: Here, we describe the first study to use MPI to detect and track DC in a migration model with immunotherapeutic applications. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for any pre-clinical study using a highly concentrated tracer bolus that shadows nearby lower signals.

Preconditioning Human Adipose-Derived Stromal Cells on Decellularized Adipose Tissue Scaffolds Within a Perfusion Bioreactor Modulates Cell Phenotype and Promotes a Pro-regenerative Host Response.

Cell-based therapies involving the delivery of adipose-derived stromal cells (ASCs) on decellularized adipose tissue (DAT) scaffolds are a promising approach for soft tissue augmentation and reconstruction. Our lab has recently shown that culturing human ASCs on DAT scaffolds within a perfusion bioreactor prior to implantation can enhance their capacity to stimulate in vivo adipose tissue regeneration. Building from this previous work, the current study investigated the effects of bioreactor preconditioning on the ASC phenotype and secretory profile in vitro, as well as host cell recruitment following implantation in an athymic nude mouse model. Immunohistochemical analyses indicated that culturing within the bioreactor increased the percentage of ASCs co-expressing inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), as well as tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10), within the peripheral regions of the DAT relative to statically cultured controls. In addition, bioreactor culture altered the expression levels of a range of immunomodulatory factors in the ASC-seeded DAT. In vivo testing revealed that culturing the ASCs on the DAT within the perfusion bioreactor prior to implantation enhanced the infiltration of host CD31+ endothelial cells and CD26+ cells into the DAT implants, but did not alter CD45+F4/80+CD68+ macrophage recruitment. However, a higher fraction of the CD45+ cell population expressed the pro-regenerative macrophage marker CD163 in the bioreactor group, which may have contributed to enhanced remodeling of the scaffolds into host-derived adipose tissue. Overall, the findings support that bioreactor preconditioning can augment the capacity of human ASCs to stimulate regeneration through paracrine-mediated mechanisms.

Putative Concussion Biomarkers Identified in Adolescent Male Athletes Using Targeted Plasma Proteomics.

Sport concussions can be difficult to diagnose and if missed, they can expose athletes to greater injury risk and long-lasting neurological disabilities. Discovery of objective biomarkers to aid concussion diagnosis is critical to protecting athlete brain health. To this end, we performed targeted proteomics on plasma obtained from adolescent athletes suffering a sports concussion. A total of 11 concussed male athletes were enrolled at our academic Sport Medicine Concussion Clinic, as well as 24 sex-, age- and activity-matched healthy control subjects. Clinical evaluation was performed and blood was drawn within 72 h of injury. Proximity extension assays were performed for 1,472 plasma proteins; a total of six proteins were considered significantly different between cohorts (P < 0.01; five proteins decreased and one protein increased). Receiver operating characteristic curves on the six individual protein biomarkers identified had areas-under-the-curves (AUCs) for concussion diagnosis ≥0.78; antioxidant 1 copper chaperone (ATOX1; AUC 0.81, P = 0.003), secreted protein acidic and rich in cysteine (SPARC; AUC 0.81, P = 0.004), cluster of differentiation 34 (CD34; AUC 0.79, P = 0.006), polyglutamine binding protein 1 (PQBP1; AUC 0.78, P = 0.008), insulin-like growth factor-binding protein-like 1 (IGFBPL1; AUC 0.78, P = 0.008) and cytosolic 5'-nucleotidase 3A (NT5C3A; AUC 0.78, P = 0.009). Combining three of the protein biomarkers (ATOX1, SPARC and NT5C3A), produced an AUC of 0.98 for concussion diagnoses (P < 0.001; 95% CI: 0.95, 1.00). Despite a paucity of studies on these three identified proteins, the available evidence points to their roles in modulating tissue inflammation and regulating integrity of the cerebral microvasculature. Taken together, our exploratory data suggest that three or less novel proteins, which are amenable to a point-of-care immunoassay, may be future candidate biomarkers for screening adolescent sport concussion. Validation with protein assays is required in larger cohorts.

Infiltrating Hematogenous Macrophages Aggregate Around ß-Amyloid Plaques in an Age- and Sex-Dependent Manner in a Mouse Model of Alzheimer Disease.

ß-Amyloid (Aß) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMϕ) from hematogenous macrophages (hMϕ), as they are morphologically and phenotypically similar when activated. To distinguish between mMϕ and hMϕ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMϕ, but not in mMϕ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMϕ from hMϕ. Mutant mice demonstrated robust increases in Aß1-42, area of Aß plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aß accumulation, paralleled by the accumulation of hMϕ around Aß plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aß plaques was age- and sex-dependent and preceded cognitive impairment.

Fluorine-19 Cellular MRI Detection of In Vivo Dendritic Cell Migration and Subsequent Induction of Tumor Antigen-Specific Immunotherapeutic Response.

PURPOSE: A major hurdle in the advancement of cell-based cancer immunotherapies is the inability to track in vivo therapeutic cell migration. With respect to dendritic cell (DC)-based cancer immunotherapies, this lack of knowledge represents an even greater hurdle as the quantity of tumor-antigen specific DC reaching a secondary lymphoid organ post injection is predictive of the magnitude of the ensuing tumor-specific immune response. We propose fluorine-19 (F-19) cellular magnetic resonance imaging (MRI) as a suitable and non-invasive imaging modality capable of detecting and quantifying DC migration in vivo and thus, serving as a surrogate marker of DC-based immunotherapeutic effectiveness. PROCEDURES: Murine DC were generated from bone marrow precursors and labeled with a [19F]perfluorocarbon ([19F]PFC)-based cell labeling agent. DC were characterized by viability and phenotyping assessments as well as characterized by ability to induce in vivo tumor-specific immune responses following immunization in a B16-F10 mouse model of melanoma. The in vivo migration of [19F]PFC (PFC)-labeled DC was first compared to control unlabeled DC by microscopy and then measured using F-19 cellular MRI. RESULTS: Culture conditions were optimized such that > 90 % of DC labeled with PFC without affecting viability, phenotype, and function. This optimization permitted consistent detection of PFC-labeled DC migration using F-19 cellular MRI and resulted in the first successful comparison of in vivo migration between PFC-labeled and control unlabeled therapeutic cells of the same origin. PFC-labeled DC are migration-competent in vivo in a B16-F10 tumor-bearing mouse model. CONCLUSIONS: We report a non-invasive and longitudinal imaging modality capable of detecting and quantifying therapeutic cell migration at both 9.4 and 3 Tesla (T) and suitable for therapeutic cell tracking in a tumor-bearing mouse model. F-19 MRI cell tracking is broadly applicable across disease states and is conducive to clinical translation.

Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages.

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells.

BACKGROUND: Adverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells. Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome. RESULTS: Redox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines. CONCLUSION: SMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR.

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting.

A 19Fluorine (19F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19F cell-labeling agent without affecting functionality or affecting viability. The 19F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1H/19F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19F-labeled PBMC using clinical MRI protocols. Thus, 19F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Tn-MUC1 DC Vaccination of Rhesus Macaques and a Phase I/II Trial in Patients with Nonmetastatic Castrate-Resistant Prostate Cancer.

MUC1 is a glycoprotein expressed on the apical surface of ductal epithelial cells. Malignant transformation results in loss of polarization and overexpression of hypoglycosylated MUC1 carrying truncated carbohydrates known as T or Tn tumor antigens. Tumor MUC1 bearing Tn carbohydrates (Tn-MUC1) represent a potential target for immunotherapy. We evaluated the Tn-MUC1 glycopeptide in a human phase I/II clinical trial for safety that followed a preclinical study of different glycosylation forms of MUC1 in rhesus macaques, whose MUC1 is highly homologous to human MUC1. Either unglycosylated rhesus macaque MUC1 peptide (rmMUC1) or Tn-rmMUC1 glycopeptide was mixed with an adjuvant or loaded on autologous dendritic cells (DC), and responses were compared. Unglycosylated rmMUC1 peptide induced negligible humoral or cellular responses compared with the Tn-rmMUC1 glycopeptide. Tn-rmMUC1 loaded on DCs induced the highest anti-rmMUC1 T-cell responses and no clinical toxicity. In the phase I/II clinical study, 17 patients with nonmetastatic castrate-resistant prostate cancer (nmCRPC) were tested with a Tn-MUC1 glycopeptide-DC vaccine. Patients were treated with multiple intradermal and intranodal doses of autologous DCs, which were loaded with the Tn-MUC1 glycopeptide (and KLH as a positive control for immune reactivity). PSA doubling time (PSADT) improved significantly in 11 of 16 evaluable patients (P = 0.037). Immune response analyses detected significant Tn-MUC1-specific CD4+ and/or CD8+ T-cell intracellular cytokine responses in 5 out of 7 patients evaluated. In conclusion, vaccination with Tn-MUC1-loaded DCs in nmCRPC patients appears to be safe, able to induce significant T-cell responses, and have biological activity as measured by the increase in PSADT following vaccination. Cancer Immunol Res; 4(10); 881-92. ©2016 AACR.

CD11d integrin blockade reduces the systemic inflammatory response syndrome after traumatic brain injury in rats.

Traumatic CNS injury triggers a systemic inflammatory response syndrome (SIRS), in which circulating inflammatory cells invade body organs causing local inflammation and tissue damage. We have shown that the SIRS caused by spinal cord injury is greatly reduced by acute intravenous treatment with an antibody against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocyte/macrophages, a treatment that reduces their efflux from the circulation. Traumatic brain injury (TBI) is a frequently occurring injury after motor vehicle accidents, sporting and military injuries, and falls. Our studies have shown that the anti-CD11d treatment diminishes brain inflammation and oxidative injury after moderate or mild TBI, improving neurological outcomes. Accordingly, we examined the impact of this treatment on the SIRS triggered by TBI. The anti-CD11d treatment was given at 2h after a single moderate (2.5-3.0 atm) or 2 and 24h after each of three consecutive mild (1.0-1.5 atm) fluid percussion TBIs. Sham-injured, saline-treated rats served as controls. At 24h, 72 h, and 4 or 8 weeks after the single TBI and after the third of three TBIs, lungs of rats were examined histochemically, immunocytochemically and biochemically for downstream effects of SIRS including inflammation, tissue damage and expression of oxidative enzymes. Lung sections revealed that both the single moderate and repeated mild TBI caused alveolar disruption, thickening of inter-alveolar tissue, hemorrhage into the parenchyma and increased density of intra-and peri-alveolar macrophages. The anti-CD11d treatment decreased the intrapulmonary influx of neutrophils and the density of activated macrophages and the activity of myeloperoxidase after these TBIs. Moreover, Western blotting studies showed that the treatment decreased lung protein levels of oxidative enzymes gp91(phox), inducible nitric oxide synthase and cyclooxygenase-2, as well as the apoptotic pathway enzyme caspase-3 and levels of 4-hydroxynonenal-bound proteins (an indicator of lipid peroxidation). Decreased expression of the cytoprotective transcription factor Nrf2 reflected decreased lung oxidative stress. Anti-CD11d treatment also diminished the lung concentration of free radicals and tissue aldehydes. In conclusion, the substantial lung component of the SIRS after single or repeated TBIs is significantly decreased by a simple, minimally invasive and short-lasting anti-inflammatory treatment.

G-CSF preferentially supports the generation of gut-homing Gr-1high macrophages in M-CSF-treated bone marrow cells.

The G-CSF is best known for its activity in the generation and activation of neutrophils. In addition, studies on G-CSF(-/-) or G-CSFR(-/-) mice and BMC cultures suggested a role of G-CSF in macrophage generation. However, our understanding on the role of G-CSF in macrophage development is limited. Here, using in vitro BMC models, we demonstrated that G-CSF promoted the generation of Gr-1(high)/F4/80(+) macrophage-like cells in M-BMCs, likely through suppressing cell death and enhancing generation of Gr-1(high)/F4/80(+) macrophage-like cells. These Gr-1(high) macrophage-like cells produced "M2-like" cytokines and surface markers in response to LPS and IL-4/IL-13, respectively. Adoptive transfer of EGFP-expressing (EGFP(+)) M-BMCs showed a dominant, gut-homing phenotype. The small intestinal lamina propria of G-CSFR(-/-) mice also harbored significantly reduced numbers of Gr-1(high)/F4/80(+) macrophages compared with those of WT mice, but levels of Gr-1(+)/F4/80(-) neutrophil-like cells were similar between these mice. Collectively, these results suggest a novel function of G-CSF in the generation of gut-homing, M2-like macrophages.

Engineering lentiviral vectors for modulation of dendritic cell apoptotic pathways.

BACKGROUND: Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. Unfortunately, cancer cells can often disarm differentiated DCs by rendering them incapable of maturation or by promoting their apoptosis. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. In addition to undergoing cancer-induced apoptosis, it is well established that DCs are intrinsically short-lived cell types. It is likely that a significant portion of infused DCs undergo apoptosis prior to locating and activating naïve TAA-reactive T cells. METHODS: In our current study, we constructed and investigated novel bicistronic lentivectors (LVs) encoding the cDNA for the xeno-TAA, rat HER-2/neu (rHER-2), along with five candidate mouse DC survival factors (c-FLIPS, c-FLIPL, Bcl-XL, M11L, and AKT-1) that operate in both the extrinsic and intrinsic cycles of apoptosis. The murine DC cell line, DC2.4 was transduced separately with each novel LV construct. Infected cells were enriched via flow cytometric methods based on rHER-2 expression. Transduced DC2.4 cell lines were then exposed to Fetal Calf Serum (FCS) withdrawal and to specific pharmacological apoptosis-inducing agents. DC2.4 cell death was assayed based on Annexin V and PI double-positive staining via flow cytometry. The phenotype and function of transduced DC2.4 cells and primary bone marrow-derived DCs were then assessed via expression and secretion of DC markers and cytokines, respectively. RESULTS: DC2.4 cells transduced with LVs encoding cDNAs for c-FLIPS, c-FLIPL, Bcl-XL, and M11L were protected from apoptosis when exposed to low FCS-containing culture media. When treated with an anti-CD95 antibody, only DC2.4 cells transduced with LVs encoding c-FLIPS and c-FLIPL were protected from apoptosis. In contrast, only DC2.4 cells transduced with LVs encoding Bcl-XL and M11L were protected from effects of staurosporine (STS) treatment. Also, LV-modified DCs maintained their original phenotype and function. CONCLUSIONS: We present evidence that by employing novel recombinant bicistronic LVs we can simultaneously load DCs with a relevant TAA and block apoptosis; thereby confirming the usage of such LVs in the modulation of DC lifespan and function.

Temporal changes in monocyte and macrophage subsets and microglial macrophages following spinal cord injury in the Lys-Egfp-ki mouse model.

The role of hematogenous (hMΦ) and microglial (mMΦ) macrophages following spinal cord injury (SCI) remains unclear as they are not distinguished easily from each other in the lesion area. We have recently described the temporal and spatial response to SCI of each MΦ population using the lys-EGFP-ki mouse that enables EGFP(+) hMΦ to be distinguished from EGFP(-) mMΦ at the lesion site. In the present study, we characterized the response of monocyte and hMΦ subsets and mMΦ to SCI. We describe, for the first time, the responses of circulating classical (pro-inflammatory) and non-classical monocyte subsets to SCI. Additionally, we show the presence of classical and non-classical hMΦ at the SCI lesion. Importantly, we demonstrate that the 'classical pro-inflammatory' hMΦ respond in the acute (1d, 3d) stages of SCI while the 'non-classical' hMΦ respond in the sub-acute (7d, 14d) phase of SCI. At later time points (6weeks post injury) classical hMΦ return to the injury site. Our study offers new insight into the cellular inflammatory response that occurs after SCI and suggests that the timing and targets of anti-inflammatory therapies may be crucial to maximize neuroprotection at the acute and more chronic stages of SCI.

Tracking and evaluation of dendritic cell migration by cellular magnetic resonance imaging.

Cellular magnetic resonance imaging (MRI) is a means by which cells labeled ex vivo with a contrast agent can be detected and tracked over time in vivo. This technology provides a noninvasive method with which to assess cell-based therapies in vivo. Dendritic cell (DC)-based vaccines are a promising cancer immunotherapy, but its success is highly dependent on the injected DC migrating to a secondary lymphoid organ such as a nearby lymph node. There the DC can interact with T cells to elicit a tumor-specific immune response. It is important to verify DC migration in vivo using a noninvasive imaging modality, such as cellular MRI, so that important information regarding the anatomical location and persistence of the injected DC in a targeted lymph node can be provided. An understanding of DC biology is critical in ascertaining how to label DC with sufficient contrast agent to render them detectable by MRI. While iron oxide nanoparticles provide the best sensitivity for detection of DC in vivo, a clinical grade iron oxide agent is not currently available. A clinical grade (19) Fluorine-based perfluorcarbon nanoemulsion is available but is less sensitive, and its utility to detect DC migration in humans remains to be demonstrated using clinical scanners presently available. The ability to quantitatively track DC migration in vivo can provide important information as to whether different DC maturation and activation protocols result in improved DC migration efficiency which will determine the vaccine's immunogenicity and ultimately the tumor immunotherapy's outcome in humans.

Insights into origins of Human T-cell Lymphotropic Virus Type 1 based on new strains from aboriginal people of Canada.

The causes of the worldwide distribution of Human T-cell Lymphotropic Virus Type 1 (HTLV-1) remain incompletely understood, with competing hypotheses regarding the number and timing of events leading to intercontinental spread on historical and prehistoric timescales. Ongoing discovery of this virus in aboriginal populations of Asia and the Americas has been the main source of evidence for the latter. We conducted molecular phylogenetic and dating analyses for 13 newly reported HTLV-1 strains from Canada. We analyzed two full-length proviral genomes from aboriginal residents of Nunavut (an autonomous territory in Northern Canada including most of the Canadian Arctic), 11 long-terminal-repeat (LTR) sequences from aboriginal residents of British Columbia's Pacific coast, and 2 LTR sequences from non-aboriginal Canadians. Phylogenetic analysis demonstrated a well-supported affinity between the two Nunavut strains and two East Asian strains, suggesting the presence of an Asian-American sublineage within the widespread "transcontinental" subgroup A clade of HTLV-1 Cosmopolitan subtype a. This putative sublineage was estimated to be 5400-11,900 years in age, consistent with a long-term presence of HTLV-1 in aboriginal populations of the Canadian Arctic. Phylogenetic affinities of the other 11 Canadian HTLV-1 aboriginal strains were diverse, strengthening earlier evidence for multiple incursions of this virus into coastal aboriginal populations of British Columbia. Our results are consistent with the hypothesis of ancient presence of HTLV-1 in aboriginal populations of North America.

Differential detection and distribution of microglial and hematogenous macrophage populations in the injured spinal cord of lys-EGFP-ki transgenic mice.

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

Cellular MRI as a suitable, sensitive non-invasive modality for correlating in vivo migratory efficiencies of different dendritic cell populations with subsequent immunological outcomes.

The clinical application of dendritic cells (DC) as adjuvants in immunotherapies such as the cell-based cancer vaccine continues to gain interest. The overall efficacy of this emerging immunotherapy, however, remains low. Studies suggest the stage of maturation and activation of ex vivo-prepared DC immediately prior to patient administration is critical to subsequent DC migration in vivo, which ultimately affects overall vaccine efficacy. While it is possible to generate mature and activated DC ex vivo using various stimulatory cocktails, in the case of cancer patients, the qualitative and quantitative assessment of which DC stimulatory cocktail works most effectively to enhance subsequent DC migration in vivo is difficult. Thus, a non-invasive imaging modality capable of monitoring the real-time migration of DC in long-term studies is required. In this paper, we address whether cellular magnetic resonance imaging (MRI) is sufficiently sensitive to quantitatively detect differences in the migratory abilities of two different DC preparations: untreated (resting) versus ex vivo matured in a mouse model. In order to distinguish our ex vivo-generated DC of interest from surrounding tissues in magnetic resonance (MR) images, DC were labeled in vitro with the superparamagnetic iron oxide (SPIO) nanoparticle FeREX®. Characterization of DC phenotype and function following addition of a cytokine maturation cocktail and the toll-like receptor ligand CpG, both in the presence and in the absence of SPIO, were also carried out. Conventional histological techniques were used to verify the quantitative data obtained from MR images. This study provides important information relevant to tracking the in vivo migration of ex vivo-prepared and stimulated DC.