CD4 Receptor is a Key Determinant of Divergent HIV-1 Sensing by Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDC) are innate immune cells that sense viral nucleic acids through endosomal Toll-like receptor (TLR) 7/9 to produce type I interferon (IFN) and to differentiate into potent antigen presenting cells (APC). Engagement of TLR7/9 in early endosomes appears to trigger the IRF7 pathway for IFN production whereas engagement in lysosomes seems to trigger the NF-κB pathway for maturation into APC. We showed previously that HIV-1 (HIV) localizes predominantly to early endosomes, not lysosomes, and mainly stimulate IRF7 rather than NF-κB signaling pathways in pDC. This divergent signaling may contribute to disease progression through production of pro-apoptotic and pro-inflammatory IFN and inadequate maturation of pDCs. We now demonstrate that HIV virions may be re-directed to lysosomes for NF-κB signaling by either pseudotyping HIV with influenza hemagglutinin envelope or modification of CD4 mediated-intracellular trafficking. These data suggest that HIV envelope-CD4 receptor interactions drive pDC activation toward an immature IFN producing phenotype rather than differentiation into a mature dendritic cell phenotype.

Methods of peripheral nerve tissue preparation for second harmonic generation imaging of collagen fibers.

Second harmonic generation (SHG) imaging of the peripheral nerve using multi-photon microscopy is a novel technique with little documentation. It affords the significant possibility of non-destructive imaging of internal nerve anatomy. The nature of nerve tissue, especially its size and viscoelastic properties, present special challenges for microscopy. While nerves are under an innate in situ strain, they retract once dissected, thus distorting microscopic structure. The challenge is to preserve the nerve in its natural strain range to obtain images that most truly reveal its structure. This study examined backscattered SHG images of rat median nerve prepared by several different methods to compare image quality and content. Nerve segments were fixed under strained (constant load or length) and unstrained conditions and imaged as whole nerve as well as plastic (methyl methacrylate) and paraffin embedded sections. These were tested for optimal excitation wavelength, quantitative image contrast, and overall quality. Root mean squared (RMS) contrast proved to be a reliable measure of the level of image contrast perceived by eye. We concluded that images obtained from tissue sections (plastic and paraffin) provided the most accurate and revealing SHG images of peripheral nerve structure. Removing the embedding material prior to imaging significantly improved image quality. Optimal excitation wavelengths were consistent regardless of the preparation method.

Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration.

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro, and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS.

Development of an automated gamma-H2AX immunocytochemistry assay.

gamma-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify gamma-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for gamma-H2AX focus formation by immunofluorescence analysis. The gamma-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and gamma-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r(2) correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when gamma-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of gamma-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify gamma-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.

Real-time diagnostic imaging of tumors and metastases by use of a replication-competent herpes vector to facilitate minimally invasive oncological surgery.

Current efforts on expanding minimally invasive techniques into the realm of oncological surgery are hindered by lack of accurate visualization of tumor margins and failure to detect micro metastases in real time. We used a systemic delivery of a herpes viral vector with cancer-selective infection and replication to precisely differentiate between normal and malignant tissue. NV1066 is a genetically modified, replication-competent herpes simplex virus carrying a transgene for enhanced green fluorescent protein (GFP). We tested the potential of NV1066 in delineating tumor tissue in vitro and in vivo in a wide range of cancers and whether NV1066-induced GFP expression can detect small foci of tumors and metastases in in vivo models using an operating endoscope with fluorescent filters. Our findings indicate that NV1066 can be used for real-time intraoperative imaging and enhanced detection of early cancers and metastases. We demonstrate that a single dose of NV1066, administered either locally (intratumoral or intracavitary) or systemically, will detect loco-regional and distant disease throughout the body. Such cancer selectivity is confirmed in 110 types of cancer cells from 16 different primary organs. Fluorescence-aided minimally invasive endoscopy revealed microscopic tumor deposits unrecognized by conventional laparoscopy/thoracoscopy. Furthermore, NV1066 ability to transit and infect tumor and metastases is proven in syngenic and transplanted tumors in different animal models, both immunocompetent and immunodeficient. Cancer-selective GFP expression is confirmed by histology, immunohistochemistry, and qRT-PCR. These studies form the basis for real-time, intraoperative diagnostic imaging of tumor and metastases by minimally invasive endoscopic technology.

Estrogen enhances the efficacy of an oncolytic HSV-1 mutant in the treatment of estrogen receptor-positive breast cancer.

Oncolytic herpes simplex virus-1 (HSV-1) mutants selectively replicate in and lyse tumor cells. Viral replication is dependent on the cellular proliferative mechanism. Estrogen increases cellular proliferation and decreases apoptosis in estrogen receptor-positive (ER+) human breast cancer cells. We hypothesize that the cellular changes produced by estrogen may enhance oncolytic viral replication and improve the treatment of ER+ breast cancer cells. Estrogen increased proliferation and replication of the HSV-1 mutant, NV1066, in ER+ breast cancer cells. Additionally, cells grown with estrogen had lower rates of apoptosis and higher bcl-2 levels at baseline and after infection. Estrogen enhanced the oncolytic effect of NV1066, with cell kills of 95% and 97% at MOIs of 0.1 and 0.5, compared to 53 and 87% respectively without estrogen (p<0.001). Therapy of ER+ human breast cancer cells with a replication-competent HSV-1 mutant is improved in the presence of estrogen, in contrast to more standard therapies, such as chemotherapy and radiation, which demonstrate decreased efficacy in similar conditions. These data provide the mechanistic basis for the use of oncolytic HSV-1 in patients with hormone receptor-positive breast cancer, particularly if the disease progresses with conventional therapies.

Virally directed fluorescent imaging improves diagnostic sensitivity in the detection of minimal residual disease after potentially curative cytoreductive surgery.

Completeness of cytoreduction is an independent prognostic factor after cure-intended surgery for peritoneal carcinomatosis. NV1066, a genetically engineered herpes simplex virus carrying the transgene for green fluorescent protein, selectively infects cancer cells. We sought to determine the feasibility of virally directed fluorescent imaging in the intraoperative detection of minimal residual disease after cytoreductive surgery. NV1066 infected human gastric cancer cells, OCUM-2MD3, and mesothelioma JMN cells at all doses. The infected cells expressed green fluorescent protein and were killed. OCUM-2MD3, and mesothelioma JMN cells at all doses. Peritoneal carcinomatosis was established in mice by injection of OCUM cells into the peritoneal cavity. Forty-eight hours after intraperitoneal injection of NV1066, two experienced surgeons resected all visible disease and identified mice free of disease. Eight of 13 mice thought to be free of disease were found to have residual disease as identified by green fluorescence (mean number of observations: 5; range: 1-9). Residual disease was most frequently observed in the retroperitoneum, pelvis, peritoneal surface, and liver. Specificity of NV1066 infection to tumor nodules was confirmed by immunohistochemistry and by polymerase chain reaction for viral gene. Virally directed fluorescent imaging, a novel molecular imaging technology, can be used for real-time visualization of minimal residual disease after cytoreductive surgery and can improve the completeness of cure-intended resection.

Second harmonic generation imaging and Fourier transform spectral analysis reveal damage in fatigue-loaded tendons.

Conventional histologic methods provide valuable information regarding the physical nature of damage in fatigue-loaded tendons, limited to thin, two-dimensional sections. We introduce an imaging method that characterizes tendon microstructure three-dimensionally and develop quantitative, spatial measures of damage formation within tendons. Rat patellar tendons were fatigue loaded in vivo to low, moderate, and high damage levels. Tendon microstructure was characterized using multiphoton microscopy by capturing second harmonic generation signals. Image stacks were analyzed using Fourier transform-derived computations to assess frequency-based properties of damage. Results showed 3D microstructure with progressively increased density and variety of damage patterns, characterized by kinked deformations at low, fiber dissociation at moderate, and fiber thinning and out-of-plane discontinuities at high damage levels. Image analysis generated radial distributions of power spectral gradients, establishing a "fingerprint" of tendon damage. Additionally, matrix damage was mapped using local, discretized orientation vectors. The frequency distribution of vector angles, a measure of damage content, differed from one damage level to the next. This study established an objective 3D imaging and analysis method for tendon microstructure, which characterizes directionality and anisotropy of the tendon microstructure and quantitative measures of damage that will advance investigations of the microstructural basis of degradation that precedes overuse injuries.

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.