Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an  Alpha-herpesviruses, including human herpes simplex virus 1 and 2, varicella zoster virus and swine pseudorabies virus, infect the peripheral nervous system of their hosts. Symptoms often include itching, numbness, or pain. Understanding of the physiological basis for these characteristic sensory and motor anomalies remains limited. To provide more insight, we examined the electrical activity of infected neurons in culture. We used pseudorabies virus (PRV) because infected animals show strain dependent, and often, dramatic symptoms (violent scratching and self mutilation). We found that infected neurons exhibited increased action potential (AP) rates. Infected neurons also became electrically coupled, proceeding from small molecular weight dye sharing and coupled activity to large molecular weight dye sharing and complete electrical synchrony. Late in infection, cell bodies fused to form syncytia. When neurons were infected with a virulent strain that could not express glycoprotein B, a member of the viral membrane fusion complex, AP rates were not increased. Changes in electrical connectivity and dye transfer were not observed, and syncytia did not occur. These results suggest that infection induced elevated activity and electrical coupling result from virally induced membrane fusion and may underlie neurological symptoms observed during infection. Alpha-herpesviruses infect a wide variety of cell types, but their hallmark is infection of the nervous systems of their hosts. Infections result in a variety of symptoms, some of which suggest an effect on motor and sensory neuron activity. For example, herpes simplex virus type 1 causes herpes labialis with the sensations of numbness and tingling Pseudorabies virus (PRV) is an alpha-herpesvirus with a broad host range that is the causative agent of Aujesky's disease in adult swine. All other susceptible animals  typically die following infection preceded by marked symptoms of PNS and central nervous system (CNS) dysfunction. Symptoms occur in a strain dependent manner and include severe pruritus with frantic self-mutilation (known as the “mad itch”), loss of motor coordination, and ataxia Several early studies examined the effects of virulent PRV infection on the electrical function of infected neurons of non-natural hosts Because of their broad host range and reduced virulence, selected attenuated strains of PRV are used to trace chains of connected neurons in a variety of non-native hosts In this study we describe the effects of infection of virulent and attenuated strains of PRV on neuronal activity and connectivity using whole-cell patch recording methods on dissociated primary cultures of embryonic rat SCG neurons. The PRV strains used included a virulent PRV Becker strain expressing GFP (PRV 151), an avirulent mutant derivative of PRV Becker (PRV 233) that cannot express gB, an essential component of the viral membrane fusion complex We conclude that the viral membrane fusion complex  produces fusion pores several hours after infection that enable ions to flow between neurons resulting in electrical  coupling. At early times, the pores are small, open to diffusion of small molecular weight dyes such as Lucifer Yellow. These pores effectively couple the electrical activity of neurons. At later times, pores are larger, open to diffusion of larger molecular weight rhodamine-dextran conjugates. These larger pores completely synchronize firing events between involved neurons. The delay, but not elimination, of all firing and coupling events in PRV 152 infected neurons implies that mutations in the PRV Bartha genome modulate action or localization of the viral membrane fusion complex in neurons.in situ and patch clamp recordings in brain slices of infected animals. In these studies, it was difficult to determine if and when a recorded neuron was infected. To overcome these limitations, we used cultures of dissociated rat sympathetic ganglion (SCG) neurons, free of replicating epithelial and support cells. After one week in culture,  and compared their AP firing rates with that observed under mock-infection. Mock-infected SCG neurons had a mean AP firing rate of 0.3±0.2 Hz . This saIn addition to elevated AP firing rates, PRV infected neurons showed brief sub-threshold depolarizations similar to spikelets observed in electrically coupled neurons Current injections through the patch electrode were used to examine if PRV 151 infection affected the relationship of steady state voltage and AP firing rate of SCG neurons. In mock-infected neurons, AP firing rate could be elevated by injection of depolarizing currents . A mean By 8–10 hpi, PRV 151 infected neurons with elevated AP and spikelet-like event rates had a weaker response to current injections: hyperpolarizing current typically was unable to silence the neuron . As a reAt 14–16 hpi, PRV 151 infected neurons showed no relationship between AP firing rate and steady state voltage achieved by current injection . In a feBy 24–26 hpi, PRV 151 neurons showed AP firing rates that were largely independent of injected current over a broad range , neurons infected with PRV 151 had resting potentials and AP shapes comparable to mock-uninfected neurons. By 14–16 hpi, despite high firing frequency, resting potentials of PRV 151 infected neurons were strongly hyperpolarized compared to uninfected control neurons . Infected neurons remained significantly hyperpolarized until at least 24 hpi . AdditioThe shape of spontaneous APs also was changed by PRV 151 infection. AP shape was quantified by measuring the AP amplitude (difference in voltage between the absolute peak of AP), AHP amplitude  and total AP height (difference between the absolute peak of AP and the voltage 11 ms after AP peak) . During Since these changes in shape coincided with marked hyperpolarization, we explored whether hyperpolarization alone was responsible for the change in shape by injecting hyperpolarizing and depolarizing current. As seen in In addition, the AP shape at threshold of AP initiation had a generally sharper inflection in infected neurons. This change is evident by inspection of the right versus left traces in The shapes and characteristics of the spikelet-like events were suggestive of electrical coupling analogous to that observed with gap junction mediated electrical synapses between neurons. We used well characterized dye transfer methods used to characterize gap junctions For Lucifer Yellow (LY) dye transfer measurements, whole cell recordings using dye-filled electrodes were obtained from randomly chosen cell bodies at various time points after infection. Recordings were maintained for 10–15 minutes to allow dye to diffuse into the cell body from the electrode. The electrode was then removed and a one-hour incubation period was provided to allow time for LY to diffuse into its processes and to any connected neurons. Because LY fluorescence is not easily distinguished from GFP, we used the parent strain PRV Becker, which exhibits identical electrophysiological changes identical to those induced by PRV 151 infection (data not shown).When mock-infected cell bodies were filled with LY containing electrodes, cell bodies and axonal processes  were filled with dye, but no other cell bodies or processes were filled . SimilarBy measuring transfer of large molecular weight dyes (Texas-red dextran conjugates) known to be too large to pass between gap junctions , we observed that larger pores formed later in PRV 151 infection. Cell bodies were filled with various Texas-red dextran conjugates by diffusion from a patch electrode  for 10–15 minutes prior to imaging. Because Texas-red fluorescence was easily distinguished from that of GFP, PRV 151 was used in these experiments.In both mock-infected and early PRV infected (<24hrs) neurons, no transfer of the Texas red conjugates was obvious . HoweverTo relate dye transfer to electrophysiological activity, dual whole-cell patch recordings were performed on several pairs of infected neurons. As shown in At late times after infection (>18 hrs) signs of membrane fusion were visible by bright field microscopy. Specifically, membrane contours of the junction between adjacent cell bodies of some infected neurons relaxed. A distinct vertex is normally observed where the membranes of two adjacent cell bodies come into contact; however, as infection progressed, this vertex became increasingly difficult to resolve. This change suggested that cell bodies were fusing. To quantify the extent of fusion across a population of neurons in a culture dish, we examined the relationship between dye transfer and the angle at the vertex formed by the outer contours of adjacent neuronal cell bodies that either shared or did not share large MW dye. As shown in As shown above, PRV infected neurons become both electrically and dye coupled leading to the formation of cell body syncytia. Syncytia formation and cell-cell fusion are well known to occur in non-neuronal cells as a result of PRV infection. This process requires the viral fusion glycoproteins gB, gH, and gL Strikingly, gB null infected neurons showed no evidence of elevated rates of AP firing or spikelet-like events as late as 24 hpi, the last time point assayed . These iIndividual gB null PRV infected neurons showed no signs of pore formation or fusion events. Infected neurons filled with dextran conjugated Texas red (≥3000 MW) showed no dye transfer to cell bodies with closely opposing membranes  have been widely used to identify synaptically connected neurons. PRV Bartha derivatives spread only from post-synaptic to pre-synaptic neurons (retrograde spread only) and all infected animals have markedly reduced symptoms compared to virulent strains. The PRV Bartha genome carries several characterized mutations that affect anterograde spread of infection between neurons, for efficient spread of infection in the retrograde direction, and for virulence in vitro system to compare PRV 151 and PRV 152 infected neurons. SCG neurons infected with PRV 152 produced titers of infectious virus comparable to those infected with PRV 151 and the time course of infection (as measured by GFP expression) was comparable (data not shown).Given that previous reports had indicated that in animals infected with PRV 152, neurons showed no signs of abnormal electrophysiology Unlike PRV 151 infections, PRV 152 infected neurons did not show elevated rates of AP firing by 8 or 16 hours post infection . HoweverThe onset of PRV 152 spikelet-like events was also delayed by 10 hours, beginning at 14–16 hpi. By 18 hpi 66.7% of PRV 152 infected neurons showed spikelet-like events at rates greater than 10 Hz (vs. 0% PRV 151 infected), and 86.7% by 24 hpi (vs. 35.7% PRV 151). These rates were higher than those of PRV 151 infected neurons at comparable time points. However, the elevated mean AP firing rates were lower during periods in which PRV 152 infected neurons showed higher rates spikelet-like events compared to PRV 151 infected neurons. Despite this difference, the total event rate, including APs and spikelet-like events, in PRV 152 infected was not significantly different compared to PRV 151 infected neurons at 18 or 24 hpi .AP firing rates independent of steady state voltage during current injection was also delayed by 10 hours in those infected with PRV 152 . At 4–8 PRV 152 infected neurons also showed changes in resting potential and AP shape similar to PRV 151 infected neurons. By 14–16 hpi PRV 152 infected neurons became significantly hyperpolarized compared to mock-infected neurons  showed no evidence of LY transfer at 9–12 hpi 0/8, . By 18 hThese experiments indicate that infection with PRV Bartha delayed, but did not eliminate increased neuronal activity and coupling. Since there is no published evidence that PRV Bartha has mutations in gB or in membrane fusion activity, these results indicate that other mutations in the PRV Bartha genome modulate action or localization of the viral membrane fusion complex in neurons.Previous studies of rodents infected with PRV indicated that PNS ganglia, particularly the SCG, exhibited altered activity characterized by elevated AP firing rates. We used the same PNS neurons in primary culture to demonstrate that PRV infection strongly affects electrophysiology and connectivity. Moreover, using an avirulent (PRV 233) and an attenuated PRV strain (PRV 152), we were able to identify the primary and modulating events that lead to this altered activity. We found that both PRV 151 and PRV 152 infected neurons began to fire APs at elevated rates, but firing induced by PRV 152 was significantly delayed (at least 10 hours delayed). The shape of the induced APs for both infections was unusual and the induced spikelet-like events were clearly different from epsps characteristic of synaptic events. We found that the induced AP and spikelet-like events correlated with formation of small fusion pores between adjacent cells. These pores were revealed by dye transfer experiments and by observation of electrical coupling of fused neurons. Over the course of infection, the pores became larger as demonstrated by passage of large molecular weight Texas red dextran conjugates. When neurons were infected with the avirulent gB mutant, PRV 233, AP firing rates did not change and were indistinguishable from those seen in uninfected neurons. No changes in electrical connectivity or dye transfer were observed, and formation of multi-nucleated syncytia never occurred for as long as three days post infection.These results with the gB null mutant are consistent with the hypothesis that the primary initiator of altered firing of PRV infected neurons is the viral membrane fusion complex . This complex produces fusion pores several hours after infection that enable ions to flow between neurons resulting in electrical  coupling. At early times, the pores are small, open to diffusion of small molecular weight dyes such as Lucifer Yellow. These pores effectively couple the electrical activity of neurons. At later times, the pores are larger, open to diffusion of larger molecular weight rhodamine-dextran conjugates. These larger pores completely synchronize firing events between involved neurons. The delay, but not elimination, of all firing and coupling events in PRV 152 infected neurons implies that mutations in the PRV Bartha genome modulate action or localization of the viral membrane fusion complex in neurons. It is conceivable that the gB protein plays roles outside of the fusion complex that affect firing. Tests of the idea that the fusion complex is indeed the major initiator of firing would entail studies of gH and gL null viruses, which should behave like gB null mutants in these studies.Resting membrane potentials of PRV 151 and PRV 152 infected neurons were significantly hyperpolarized. This finding is surprising as hyperpolarization is usually associated with a decrease in firing rate. Hyperpolarization may be a homeostatic response of infected neurons to significantly increased rates of depolarizing inputs PRV 151 infected neurons show evidence of electrical coupling early by 8 hours after infection that correlate with increased AP firing and spikelet-like events. Neither AP firing or spikelet-like events could be silenced by hyperpolarizing cell body current injections and the shape of APs that recorded from cell bodies was characteristic of that of back-propagated APs, both suggesting events originated at outside, electrically coupled sites. Infected neurons showed progressively stronger electrical coupling and transfer of larger dye molecules as infection continued. Dual recordings revealed reciprocal connections early, in which APs recorded from one neuron occurred at the same time as a smaller, dampened depolarization (or spikelet-like event) occurred in the other. Later, adjacent neurons showed synchronous events later in infection, in which APs and spikelet-like event timing was identical in adjacent neurons. In addition, late in infection, cell bodies fused to form multi-nucleated syncytia. These data suggest that fusion of neuronal membranes occurs first on a small scale, possibly transiently, early at sites of membrane apposition. Later, these small pores expand, leading to more cytoplasmic continuity between neurons. Finally, late in infection, these pores expand resulting large rounded neuronal syncytia. PRV 152 infected neurons exhibit identical properties except that everything is delayed by at least 10 hours. As observed for PRV 151 infected neurons, increased firing rates are correlated with appearance of dye transfer events and electrical coupling.in vitro studies have suggested that alpha-herpesvirus infection can provoke neuronal membrane fusion. Scanning electron microscopy of virulent PRV infected rat CNS neurons in vitro showed evidence of neuronal membrane fusion Previous in vivo and in vitroChanges observed in AP firing rates, spikelet-like events, electrical coupling, dye transfer and formation of multi-nucleated syncytia after PRV infection were not seen in neurons infected with PRV 233, a gB null mutant. gB is an essential PRV membrane protein found in the virion envelope and on the plasma membranes of infected cells. gB is required as part of a viral protein complex for fusion of extracellular particles with host cell membranes and for spread of infection from one cell to another including trans-synaptic spread among neurons Others have reported that when antiviral drugs, such as Foscarnet, block PRV DNA replication, physiological properties  of cultured sensory neurons or in tissue explants are not altered Our experiments directly demonstrate the development of electrical coupling between infected neurons, while results obtained with a gB null virus emphasize the importance of fusion in inducing increased firing. But how does electrical coupling and fusion produce an increased firing rate? We suggest that AP initiation in electrically isolated axonal compartments is crucial. Three lines of evidence support this assertion. First, hyperpolarization of cell bodies by injected current did not change AP firing rate, demonstrating that AP rate is not determined by successive depolarizations of the cell body membrane potential to AP threshold. Second, very strong hyperpolarization reduces AP amplitude, revealing a reduced AP originating in electrically distant compartments, which must be axons as these cells have no dendrites in culture An important question is what induces APs in axons? More work is required to provide a definitive answer to this question but we note that the input resistance of an axon below threshold is normally very high The delayed development of increased firing rates after PRV 152 infection is of some interest because it suggests that fusion/coupling events are modulated in some way by other viral gene products or processes. We have preliminary evidence to suggest that at least two processes are involved: axonal sorting of viral proteins and virus particle formation.In work presented in the supplemental information , we firsWe and others have mapped and characterized several mutations in PRV Bartha responsible for its attenuated phenotype The PRV Bartha Us region harbors a 3.4kb deletion removing the coding sequences for viral membrane proteins gE, gI, and Us9, which are required for localization of PRV structural proteins to axons and axonal membranes. As a consequence, in PRV Bartha infections, viral membrane fusion proteins including gB, gH, and gL are not present in axons in vitro and in vivoRecently, we reported that repair of the Bartha UL21 locus with wild-type sequence increased efficiency of transneuronal spread both In work not shown, we infected neurons with PRV 4325, a recombinant PRV Bartha strain that carries the PRV Becker UL21 locus and the Becker Us region. The onset of firing and the rate was indistinguishable from PRV 151 infections. We conclude that reduced transneuronal spread (UL21 mutations) and absence of axonal localization of viral proteins (gE/gI/Us9 deletion) in PRV Bartha or PRV 152 infections are the primary contributors to the delayed firing phenotype.in vitro, studies using attenuated PRV Bartha derivatives as neuronal circuit tracers report that infected neurons in CNS tissue display electrophysiological phenotypes indistinguishable from uninfected neurons in vitro occur in vivo, as these strains are now being used to study circuit function using both electrical techniques and virally expressed functional indicators In contrast to our observations in PNS neurons in vitro and in vitro observations may simply reflect the time at which neurons were analyzed. In a multi-synaptic circuit, it is difficult to establish the precise time of infection. The properties of infected neurons reported in vivo may have been measured during the early stages of PRV infection, before extensive neuronal coupling and associated elevated rates of firing began.Because PRV 152 neurons are significantly delayed in exhibiting increased firing events, the discrepancy between in vitro system, neurons form an extensive, highly inter-connected network, free of any other cells, providing ample opportunity for fusion and AP propagation. In vivo, neurons are organized into functional circuits with varying degrees of inter-connectivity and often are ensheathed by glia, satellite cells or thick extracellular matrices In addition, differences in the architecture of the infected neuronal circuit must be considered. If viral induced fusion can occur at any site of close membrane apposition  where viral fusion proteins are present, then the extent of electrical coupling, fusion and un-attenuated AP firing will be limited by the quantity of potential fusion sites and circuit interconnectivity. In our in vivo.Alternatively, fusion and electrical coupling of infected CNS neurons may be revealed as more subtle changes in physiology, such as the synaptic integrity between connected neurons. One study by Glatzer el al. in vitro findings.The dramatic peripheral neuropathy exhibited by PRV infected animals may reflect elevated rates of AP firing and neuronal fusion in neurons that innervate the infected tissue. Virulent strains of PRV lead to violent scratching at the site and dermatome of inoculation While infection with virulent PRV Becker induces pruritus in the mouse flank model of infection, the attenuated strain PRV Bartha does not in vitro and in vivo after infection with other viruses. Neuron-microglia fusion has been shown in vivo and in vitro by a replication incompetent retrovirus in vitroNeuronal fusion has not been reported in PRV infected PNS ganglia or brain regions. However, previous assays, including electron microscopy, immuno-labeling and confocal microscopy, have relied on visual methods and did not measure fusion directly in vitro observations, neuronal fusion and associated changes in activity must be considered in PRV infected PNS ganglia and CNS nuclei. Future work will be directed at determining the extent to which changes in neuronal activity and connectivity occur in these tissues and their relevance to neurological symptoms central to pathogenesis.Given our All experimental protocols related to animal use were approved by the Institutional Animal Care and Use Committee of the Princeton University Research Board under protocol number 1691 and are in accordance with the regulations of the American Association for Accreditation of Laboratory Animal Care and those in the Animal Welfare Act (public law 99–198).All strains were propagated on porcine kidney (PK15) cells at a low multiplicity of infection for 48 hours and then collected into the conditioned medium as described previously 2 at (Thermo-Scientific). Medium was replaced every 3–5 days.Embryonic rat superior cervical ganglia (SCG) were isolated from Sprague-Dawley rats  between embryonic day 16.5 and E17.5 and dissociated into individual neurons as described previously 6 plaque forming units (in 600 µl over entire 35 mm dish) and incubated for 1 hour prior to replacement with neuronal medium. Mock-infection consisted of either addition of mock inoculum described above or DMEM supplemented with 2% FBS and 1% P/S.Infection of neurons was performed as described previously 3, 0.13 NaH2PO4, 10 HEPES, 7.8 glucose, 2.5 CaCl2 and 0.6 MgSO4 (OSM 320) pH 7.2. A section of culture dish side-wall was removed by melting a small section to allow unobstructed pipette access to the culture dish. Extracellular solution was maintained at 37°C (±2°C) by placing the culture dish on a heated copper plate with supporting walls and monitored during recordings . Patch pipettes pulled  from borosilicate glass  to a resistance of 4–7MΩ were filled (mM) with intracellular solution, 140 K-gluconate, 10 HEPES, 2 MgCl2, 3 ATPNa2 and 0.3 GTPNa (OSM 275) pH 7.3 (Sigma-Aldrich). Prior to and during recordings neurons were visualized with a 40× water immersion objective (Olympus LUMPIPlanFI/IR 40× 0.80) using an Olympus BX51WI microscope. Whole-cell patches were obtained from soma of individual neurons in clusters of 2 to 8 after 1–4 GΩ seals were formed. Stable whole cell recordings were observed for 1–5 min prior to recording of spontaneous activity and resting membrane potentials with a Dagan N = 0.1 or N = 0.01 headstage and BVC700 amplifier for 2–5 minutes in current clamp mode with no holding current. Next, a series of 1s current injections (−0.15nA to 0.15nA) were delivered every 10 seconds under current clamp from resting voltage. Capacitance and series (5–40MΩs) resistance were compensated. In several occasions uncompensated series resistance was subtracted off-line prior to analysis. Recordings from cells with >10 mV baseline changes through the duration of spontaneous activity were rejected. Current injection data from neurons showing this instability were also rejected. Dual recordings were obtained using the same procedure as above with a second electrode, headstage and amplifier. Following recordings, cells were briefly viewed for GFP fluorescence to confirm infection (Olympus FITC/GFP filter cube).Whole-cell patch clamp recordings were performed at 37°C on neurons grown in culture for 10–20 days. Before recording, neuronal medium was replaced with warmed physiological extracellular solution, (mM) 146 NaCl, 4.7 KCl, 1.6 NaHCOData were obtained and digitized at 20kHz using an Axon Digidata 1233a and Clampex 9 or 1440a and Clampex 10 software (Axon/Molecular Devices). Junction potential was calculated as −17 mV using Clampex software's junction potential assistant and subtracted from all raw voltage data prior to analysis. All data were analyzed offline using custom MATLAB software.Resting voltage was determined as follows: for silent neurons (0 Hz) resting voltages were determined by averaging voltage across the entire recording, for neurons with events occurring between >0 and <1 Hz the mean voltage across the entire measurement except for times when the resting membrane potential was greater than ±SEM , and for neurons showing events >1Hz the mean voltage during of periods of steady voltage, recalculated and confirmed by manual inspection of >3 1 s intervals. Action potential firing rate was determined from whole-cell recordings of 120 seconds, defined as the number of large voltage deflections crossing a threshold (>50 mV from baseline) divided by total time. AP amplitude was defined at the peak voltage following threshold crossing minus resting voltage, AHP was defined as the difference between resting voltage (baseline) and the voltage 11 ms following AP peak. AP height was defined as the difference between AP peak and voltage 11 ms following the peak.Spikelet-like events were identified by a threshold imposed on the first derivative of the smoothed voltage trace (4–20V/s). Identified events were then manually confirmed by visual inspection based on determination of maximum rise rate and time to half decay from the peak. Rates of either event (APs and spikelet-like events) were determined as the number of events divided by total recording time analyzed. Total event rate was defined as the sum of these three classes of events. Total event rates during current injections were determined as above and reported as the ratio of event rate to the maximum event rate for each cell (up to 0.15nA). Steady state voltage during injections with <3 events was defined as the mean voltage during the last 200 ms. For those injections eliciting >3 events, steady state voltage was determined as the mean voltage during the last 200 ms excluding events.Input resistance was calculated using the linear fit of steady state resting voltages recorded during injections to current injections. AP threshold was calculated by subtracting the steady state voltage during the lowest positive current injection eliciting at least one AP. Slopes were taken over 10 mV bins to account for starting resting voltage differences between treatments. DC coupling ratios were determined by current injections into one cell body during dual recordings. Values reported are the mean voltage deflection of the second cell divided by the mean voltage deflection of the directly injected cell. All data reported as ±SEM. Student t-tests or unpaired two-tailed t-tests were used to determine significance, *P<0.05.Whole-cell patch clamp recordings were performed as above with 0.5 ug/ml neutral Texas-red Dextran conjugates at 3000, 10000 or 40000 MW (Invitrogen) or 0.05% Lucifer yellow (LY), MW 457 (Invitrogen). In LY experiments, dye was allowed to diffuse from the patch pipette into the neuron for 10–15 minutes. The pipette was then removed and the extracellular medium was replaced. After one hour, fluorescence images were taken at 40× of the filled cell body and its processes, at multiple focal planes. Resulting images were assessed for additional labeled cell bodies and contiguous labeled processes were counted. In large MW dye experiments, dye was allowed to diffuse from the patch pipette and images were recorded immediately, in most cases prior to pipette removal. Images were taken at multiple planes and assessed for additional labeled cell bodies. All images were obtained using an Olympus Bx51WI microscope by a CoolSnapcf CCD camera (Photometrics) and RS Image (Roper Scientific). Fluorescence images were acquired using automatic exposure with an intensity target of 3000, limit 1s, gain 1. Images of GFP expressed from the viral genome, Texas-red or Lucifer Yellow from patch pipette, and bright-field images using Olympus HQ:FITC or HC Red filter cubes. Fluorescence images were pseudo-colored as appropriate and enhanced for contrast at 0.5% saturated pixel intensity using ImageJ (NIH). Cell bodies were considered to have dye transferred from the filled cell body if their mean intensity was three times the standard deviation of background. Resulting images were screened onto bright field images obtained at the same focal plane using Adobe Photoshop 10.0.2), were excluded from calculation the percentage of cell bodies fused. The standard curve allowed us to determination of minimum number of nuclei (cells fused) in area measurements from random unstained fields of syncytial clusters of neurons. The total number of cell bodies counted as fused by either method was divided by the total number assayed.Percentage of fused neurons was determined by a combination of the angle measured between cell bodies and, for heavily fused cultures, by the area of large rounded neuronal syncytial. The angle between the outer edge of adjacent neurons was first determined in pairs of neurons that were tested for large MW dye coupling using ImageJ. These measurements resulted in clear separation between fused (>125 degrees) and unfused neurons Click here for additional data file.Figure S1AP and spikelet-like event responses of PRV infected neuron to cell body current injections late in PRV 151 infection. (A–D) Relationship between injected current and AP rate during current injections. The rate of APs for each current injection (between I = −0.15 and 0.14nA) was divided by the maximum event rate for that cell over all injections. Ratios were averaged and plotted against injected current. Note increasingly unchanged ratios in PRV 151 infected neurons as a function of time after infection. (E–F) Ratios of AP rate/max AP rate for individual neurons fall into several categories late in infection, leading to large error bars in A–D. (E) At 14–16 hpi, 10/15 PRV 151 infected neurons showed very little change in ratio over the range of hyperpolarizing and depolarizing currents, example neuron marked with circled #1. 5/10 showed a relationship similar to that observed at 8–10 hpi, example neuron marked with #2. F) At 24–26 hpi, PRV 151 infected neurons showed AP firing rates largely independent of current injection (5/7), examples labeled as in E. (G–J) Relationship between injected current and total (AP and spikelet-like) event rate during current injections. The rate of total APs and spikelet-like events for each current injection (between I = −0.15 and 0.14nA) was divided by the maximum event rate for that cell over all injections. Ratios were averaged and plotted against injected current. As above, note increasingly unchanged ratios in infected neurons as a function of time after infection. (B–E) Mock n = 24, 4–8 hpi PRV 151 n = 9, 8–10 hpi n = 7, 14–16 hpi n = 15, 24–26 hpi n = 7. Plots were generated using data from (0.55 MB TIF)Click here for additional data file.Figure S2Infected neurons that share large MW dyes are strongly electrically coupled. Near complete electrical coupling is seen when large MW dye is able to transfer to an adjacent cell body. Voltage response of a second neuron was divided by the first neurons response to direct current injection. Examples shown are from PRV 151 infected neurons at 18–20 hpi. (A) A pair of infected neurons with no large MW transfer shows a low level of electrical coupling. (B) A pair of infected neurons with large MW dye transfer shows a high level of electrical coupling.(0.24 MB TIF)Click here for additional data file.Figure S32, larger than maximum size of a single neuron, indicated by the dotted line, were counted as fused. Those with smaller areas adjacent to other cell bodies, were counted as not fused and those with smaller areas not adjacent to other cell bodies were not included in percentages presented in Criteria for cell body-cell body fusion. (A) Histogram of measured angles between adjacent cell bodies. Angles were measured from cell pairs in which large MW did or did not transfer, at 24 hpi with either PRV151 or PRV 152. Neuron pairs with angles less than 125° were determined not fused and pairs with larger angles were determined fused. Mock-infected neurons had a similar distribution of angles. (B) Examples of measured angles, one each of fused and not fused used in calculation of % of cell bodies fused. (C) Examples of extensive fusion of clustered neurons at 40 hpi for both PRV 151 and PRV 152 infected neurons. Blue - Hoechst, (40×). Example of two-dimensional area measured, cell indicated by an asterisk in D. To estimate the number of fused cells in a syncytia, we used the minimum number of nuclei observed for measured area to avoid over counting. (D) Standard curve constructed from two-dimensional area calculated and the number of nuclei counted. Cells with areas larger than 300 µm(1.29 MB TIF)Click here for additional data file.Figure S4AP rate responses of PRV infected neuron to cell body current injections late in PRV 152 infection. A–D) Relationship between injected current and AP rate during current injections. The rate of APs for each current injection (between I = −0.15 and 0.14nA) was divided by the maximum event rate for that cell over all injections. Ratios were averaged and plotted against injected current. Note unchanged ratios in PRV 152 infected neurons are delayed until 24–26 hpi. E–F) Ratios of AP rate/max AP rate for individual neurons fall into several categories late in infection, leading to large error bars in A–D. E) At 14–16 hpi, 13/19 PRV 152 infected neurons responded to positive current injections with increased AP firing (#3), while 6/19 showed little change in rate over the range of voltages (#4). F) By 24–26 hpi, 5/8 PRV 152 infected neurons did not respond to current injections by firing increased APs. 4/8 of these had very high levels of spikelet-like events and did not fire APs during any injection and showed no change in spikelet-rate across injections J, . 3/8 PRV(0.63 MB TIF)Click here for additional data file.Figure S5Multiple alleles of PRV Becker contribute to early onset of elevated AP firing rates. (A) Genotype maps of PRV Becker and PRV Bartha recombinants assayed in B. (B) Mean AP firing rates of infected neurons at 14–16 hpi. PRV 151 + PRV 614 n = 6, PRV 158 n = 8, PRV BaBe n = 10, PRV 327 n = 7.(0.50 MB TIF)Click here for additional data file.


The loss of tumour phospho-extracellular responsive kinase (pERK) positivity is the major treatment biomarker for mitogen-activated protein kinase/extracellular responsive kinase (MEK) inhibitors. Here, we demonstrate that there is a poor correlation between pERK inhibition and the anti-proliferative effects of MEK inhibitors in melanoma cells. We suggest that Ki67 is a better biomarker for future clinical studies. Constitutive activity of BRAF is known to be critical for the proliferation and survival of melanoma cells through the activation of the RAF/MEK/ERK mitogen activated protein (MAP) kinase pathway . In lighHuman melanoma cells were isolated and cultured as described in μM) CI-1040 or LY294002 in triplicate. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays were performed as described previously  (0.01–30 Proteins were extracted and blotted as described in Melanoma cells were prepared and stained as described previously (Comparative genomic hybridization (CGH) and mutation sequencing were performed as described previously  to 10 μM (for C8161) (50 for growth inhibition (280 nM), did not show substantial reduction in pERK phosphorylation until 3 μM U0126  . When weμM U0126 . In the μM U0126 . To explM U0126  of these melanoma lines harboured the V600E mutation in BRAF. Others have demonstrated the presence of amplifications in downstream components of the cell cycle machinery such as cyclin D1 and CDK4, which may contribute to this observed resistance . The planning of future clinical trials would likely benefit from the inclusion of both a phospho-specific signal transduction marker (such as pERK) and a proliferation marker (such as Ki67) to allow a true measure of treatment progress at the level of the tumour.


Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.Targeting viral vectors to certain tissues  This approach has indeed been reported to be successful using certain peptides in vivo and, despite the obvious importance of the question, it remains open for most of these vectors whether or not a retargeting after systemic administration in vivo occurs.Efficient and specific delivery of therapeutic genes to the tissue of interest is a paramount and so far unsolved issue in gene therapy. Among the available viral vectors for gene delivery, adeno-associated virus (AAV) has gained particular attention. The low frequency of random integration into the genome in vivo faces several hurdles which are not present in vitro and the mechanisms that determine a vectors tropism and its gene transduction properties in vivo are as yet poorly understood. While in vivo biodistribution of a vector is to a considerable part defined by clearance, its gene transduction properties are rather dependent on receptor binding, cellular uptake, nuclear transfer, and transgene expression. Thus, major hurdles for receptor targeted gene transfer in vivo are to improve specific ligand-receptor interactions under circulation conditions as well as to overcome host-anti-vector immune reactions, rapid vector clearance from the circulation by the reticuloendothelial system, and endothelial cell layers as well as the extracellular matrix acting as physical barriers in vivo biopanning of random AAV peptide libraries seems to be more appropriate to select for tissue directed gene vectors than mere tissue culture-based approaches. Among the limitations faced by in vivo AAV display library selection is the difficulty to rescue and amplify tissue-targeted library viruses for multiple selection rounds as the amplification systems used in vitro are based on adenoviral superinfection and can therefore not easily be applied in living animals.Vector targeting in vivo. We further analyzed gene transduction properties of the isolated capsid variants. While the selected vectors indeed transduced their target tissue orders of magnitude better than unselected vectors, we almost invariably observed unintended transduction of heart tissue. These results show that modification of the HSPG-binding capsid domain of AAV vectors by targeting peptide insertion is necessary but not sufficient to achieve completely tissue-specific transgene expression while this technical approach may be appropriate when expansion rather than restriction of AAV tropism to the tissue of interest is needed. Our findings therefore broaden the functional understanding of AAV-2 vectors, particularly when selected from random AAV display peptide libraries.In this study, we set out to isolate tissue-directed AAV capsids using murine breast cancer and lung tissue as prototype targets. We established a novel adenovirus-free PCR based screening approach that amplifies tissue-targeted library viruses and therefore allows for multiple AAV library screening rounds after systemic application 2/CaCl2 and 10% BSA. The cell suspension was passed through 100 µm and 40 µm cell strainers, washed twice with PBS, and cultured in Iscove's Modified Dulbecco's Medium  containing 10% fetal bovine serum, 10% horse serum, 1% penicillin/streptomycin, and 1.25 µg/ml amphotericin B (Invitrogen). All cells were cultured in a humidified atmosphere at 37°C and 5% CO2. Immunodetection using a pan-cytokeratin antibody (Sigma) revealed more than 95% of cytokeratin-positive tumor cells after 24 hours in culture.293T cells , were maintained in Dulbecco's Modified Eagle's medium  containing 1% penicillin/streptomycin solution (Invitrogen) and 10% fetal calf serum . Primary murine breast cancer cells were obtained from tumors growing in female transgenic FVB mice expressing the polyoma middle T antigen under the control of the mouse mammary tumor virus promoter www.jax.org). Starting at the age of 30 days, transgenic female mice were palpated weekly for early detection of mammary tumors. The animals were anesthetized by intraperitoneal injection of 100 mg/kg body weight 10% ketamine hydrochloride  and 5 mg/kg body weight 2% xylazine hydrochloride .All procedures involving animals were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and the German Animal Protection Code. We used a transgenic breast cancer mouse model induced by the polyoma middle T antigen (PymT) under control of the mouse mammary tumor virus promoter. The mouse strain FVB/N-TgN(MMTVPyVT)634-Mul (PymT) was purchased from Jackson Laboratory . Genotyping was performed by polymerase chain reaction (PCR) as described by Jackson Laboratory (7 AAV display peptide library (random insert introduced at position R588 VP1 capsid protein numbering) with a diversity of 2×108 random clones (determined at the cloned plasmid level) was produced using a three-step protocol as described previously in vitro biopanning (6 primary PymT breast cancer cells were incubated with the AAV library at a multiplicity of infection (MOI) of 1.000 vector genomes (vg)/cell in selection round 1, 500 vg/cell in round 2, and 100 vg/cell in round 3. After 96 hours, unbound AAV library particles were removed by 3 washing steps in PBS. Surface-bound library viruses were detached by trypsin digestion for 20 minutes and subsequent washing. Previous work had shown that this additional trypsin digest is essential to enrich internalizing clones for improved transduction of the target cells . Whole cellular DNA was extracted using the QIAamp Tissue Kit . The random oligonucleotides contained in AAV library particles internalized into tumor cells were amplified by PCR using the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ and 5′-TGATGAGAATCTGTGGAGGAG-3′. For in vivo/ex vivo biopanning of AAV peptide libraries  as described for tumor selections (BglI and cloned into the SfiI-digested pMT-202-6 library backbone plasmid E. coli DH5-α (Invitrogen) using the Gene Pulser . Randomly assigned clones were sequenced using the reverse primer 5′-CAGATGGGCCCCTGAAGGTA-3′. For production of pre-selected AAV peptide libraries, 2×108 293T cells were transfected with the library plasmids at a ratio of 25 plasmids/cell using Qiagen's PolyFect reagent. pUC18 (Invitrogen) served as carrier DNA. Two hours after transfection, 293T cells were superinfected with wild-type adenovirus type 5  at an MOI of 5 infectious particles/cell for library particle amplification. After 48 h, or when cell lysis became apparent, cells were detached from the culture dish in PBS-MK  and pooled with supernatants. AAV library particles were harvested by cell lysis via three freeze-thaw cycles. Cellular DNA was removed by incubation with benzonase (Sigma) at 50 U/ml lysate at 37°C for 30 min, followed by Ad5 inactivation at 55°C for 30 min. Viral library preparations were purified using the iodixanol gradient centrifugation method as previously described A random XRecombinant AAV (rAAV) vectors displaying selected peptide sequences were generated by cloning the oligonucleotide inserts into the pXX2-187 plasmid (a derivative of the library backbone plasmid pMT-187-0-3 without ITRs) 5′-GGCGGAGTTGTTACGACAT-3′ and the reverse primer 5′-GGGACTTTCCTACTTGGCA-3′ specific for the CMV promoter sequence. The genomic titer of AAV libraries was determined using the forward primer 5′-GCAGTATGGTGTATCTACCAA-3′ and the reverse primer 5′-GCCTGGAAGAACGCCTTGTGT-3′ specific for the AAV cap gene. Real-time PCR was done in 20 µl with 0.3 µM for each CMV primer, or 0.4 µM for each AAV primer, respectively, according to the manufacturer's protocol (Abgene). For CMV primers, annealing temperature was 64°C for 15 seconds. For AAV primers, annealing temperature was 61°C for 30 seconds. Fluorescence was measured at the end of each annealing phase. A standard curve for quantification was generated by serial dilutions of the respective vector plasmid DNA. Calculations were done using MyIQ analysis software (Bio-Rad). For quantification of vectors homing to lung tissue, 5×1010 capsid-modified rAAV-luciferase vectors were injected into the tail vein of female PycB/FVB wild-type mice (n = 3 per group). After 8 d, lung tissue was removed. Whole DNA was extracted using the DNeasy tissue kit (Qiagen) and quantified using a 2100Pro spectrophotometer . For real-time PCR, 500 ng of extracted genomic DNA were used as template to amplify vector specific DNA using CMV primers as described above. To determine the amount of circulating AAV library or wild-type viruses in the blood, 1×1010 vg were injected into the tail vein of PycB/FVB wild type mice. Blood was obtained at indicated time points and centrifuged for 2 minutes at 10,000 rpm. Cell-free serum was diluted 1∶100 in ddH2O and used as template for real-time PCR using AAV specific primer pairs as described above.The AAV capsid titers were determined as described in vitro, 2×104 cells per well were seeded in 24-well plates or 5×103 cells per well in 96-well plates and incubated with AAV-luciferase vectors at an MOI of 10,000 vg/cell for 72 h. For in vivo gene transfer, 5×1010 vg of rAAV-luciferase vectors were injected into the tail vein of anesthetized animals. After 8 or 28 days, respectively, the target tissue and representative control tissues were removed, snap frozen in liquid nitrogen, and stored at −80°C. Frozen tissue samples and cell lysates were homogenized in reporter lysis buffer  and luciferase reporter gene activity was determined in a luminometer  using Promega's luciferase assay according to the manufacturer's instructions. If required, values were normalized to protein levels in each probe determined by Bradford assay (Bio-Rad).To analyze luciferase gene transduction 3 per well in 96-well plates and transduced with rAAV-SR39 vectors at an MOI of 10,000 vg/cell. After two cycles of 10 µM ganciclovir (GCV) treatment (24 hours and 72 hours after transduction), the number of viable cells was assessed as described Cells were seeded at 5×10Statistical analysis was performed using the GraphPad Prism program 3.0 . Parametric data were analyzed by one-way analysis of variance followed by a Bonferroni post test. Non-parametric data were analyzed by a Kruskal-Wallis test followed by a Dunn's post test. p values <0.05 were considered significant.7 random AAV display peptide library in vitro along the lines of pathway A in cap gene region containing the oligonucleotide insert of AAV recovered from breast cancer cells after each round of selection was amplified by nested PCR and correct size of the amplification product was verified by agarose gel electrophoresis (data not shown). The insert was cloned back into the library backbone plasmid pMT202-6 and the diversity of transformed library plasmids was at least 1×105 clones for such secondary libraries in this and subsequent selections (data not shown). New pre-selected AAV particle libraries were obtained by transfection of 293T cells with the generated secondary plasmid library in limiting dilution technique (25 library plasmid molecules per producer cell) to minimize the production of chimeric AAV library particles or mismatch of packaged DNA and displayed peptide due to uptake of multiple library genomes in one producer cell. The titers obtained with this approach were sufficient for further selection rounds (data not shown). To increase the stringency of selection, MOIs of AAV libraries were decreased from 500 vg/cell to 100 vg/cell in rounds two and three, respectively. Sequence analysis showed enrichment of several clones after two rounds of selection compared to round 1, functionally validating our novel selection method. Peptide sequences found after round 1 were RGDLGLS, RGDMSRE, DGLGRLV, and DRSPLSL. After three rounds of selection, RGDLGLS and RGDMSRE were the dominant clones  . In contin vitro can target PymT breast tumors in vivo. AAV luciferase vectors displaying the selected peptides RGDLGLS, RGDMSRE, DGLGRLV, an unselected control peptide, or no peptide (wild-type AAV), respectively, were injected intravenously into female PymT mice bearing breast cancers. Analyses of reporter gene expression in tumor tissue revealed that none of the vectors mediated gene transduction in the tumor tissue (data not shown).We therefore investigated whether the capsid mutants selected in vivo conditions is needed to enrich library clones that are able to bind cellular receptors in tumors, penetrate the tumor tissue and are internalized into tumor cells under physiological circulation conditions after intravenous administration. But we suspected that our novel PCR-based selection of AAV libraries may not be able to distinguish between library particles successfully internalized into target cells, and non-homing particles present in the circulation if the tissue is harvested too early after injection. To minimize the amount of circulating AAV library particles in our tissue samples at the time point of harvest, we analyzed the kinetics of circulating AAV library particles. Therefore, AAV were injected intravenously at 1×1010 vg per mouse, blood samples were collected at various time points, and the amount of circulating particles in the serum was quantified by real-time PCR. Clearance rates were comparable in AAV library particles and wild-type viruses . After 4 rounds of selection, sequencing revealed the enrichment of serine and glycine-rich peptide motifs and repetition of several single clones. In particular, the motifs GGLSGXS and ESGXXXX, and the single clones EYRDSSG, QMSGGVA, EEPALRA, as well as APTLGLS were enriched during “in vivo/ex vivo” selections . Luciferase gene expression in the breast tumor tissue was evaluated 8 days after intravenous injection in tumor-bearing PymT mice in a screening experiment to assess which clones should be investigated in detail. Five of the clones  showed an increased transduction of the breast tumor tissue compared to wild-type AAV vectors, whereas unselected control vectors did not mediate any gene expression (data not shown). We chose the most promising vectors for further experiments in a larger group of animals (n = 5 mice per clone). Following intravenous injection, the selected clones transduced tumor tissue up to ∼275-fold more efficient compared to wild-type AAV vectors (in vivo gene transduction with independent vector preparations for DLGSARA and ESGLSQS precisely confirmed our results (data not shown).To assess whether the  vectors . To furt vectors . Moderatin vivo AAV library screenings depends on the target tissue the library was selected for, we also selected AAV libraries for preferential homing into lung tissue. The screening was done along the lines of the tumor targeting approach (A/S) , which was enriched independently in both selection procedures . Vectors were administered intravenously, and DNA was recovered from lung tissue after 8 days. Quantitative PCR of the CMV promoter region of the vectors revealed an up to 63-fold higher yield for the selected capsid variants compared to AAV-2 wild-type vectors and up to 74-fold higher yield compared to random control insert vectors , this issue has been addressed by the insertion of peptide ligands into the vector capsid in vivo use is very limited, especially if delivered intravenously.Vector targeting in vitroin vivo use.The screening of random AAV display peptide libraries is an innovative tool to select for vectors efficiently transducing any cell type of interest and has been described and validated for various cell types in vitro transduce the cell type they were selected on with high efficiency but fail to mediate breast cancer tissue directed gene transduction after systemic administration. As a consequence, we performed screenings of AAV libraries in vivo over multiple selection rounds after tail vein injection via the blood stream, using tumor and lung as target tissues. Several peptide clones were enriched in tumors and a clear-cut peptide sequence motif was recovered from the lung. While we achieved transduction of the target tissue by the selected vectors, we failed to achieve truly tissue-specific transgene expression. Therefore, except for liver transduction, most of our selected vectors have a tropism that is expanded to rather than specific for the tissue of interest compared to wild-type AAV-2. This may be due to several reasons: 1) the lack of tissue-specific receptors; 2) the expression of receptors conferring optimum transduction in several tissues, so capsids targeting receptors that are tissue-specific, but less efficient for transduction are not enriched; 3) superordinate (not receptor-dependent) factors influencing the selection process such as endothelial barriers, blood-derived factors, or extracellular matrix interactions. The first reason can be virtually excluded based on the overwhelming success rate of in vivo tissue targeting using phage display libraries in vivo binding behavior may have been enriched in the lung irrespective of tissue specificity due to the first-pass effect after intravenous injection. These vectors may well be cell type-specific but not tissue-specific. They may be directed to endothelia in general, which is underlined by the fact that a similar capsid mutant (PRSVTVP) has been previously selected on primary endothelial cells in vitroin vivo that can be achieved in tissues other than the lung. If the lung is the target of a screening, additional negative selection steps on endothelial cells prior to in vivo selection might alleviate some of the specificity challenges encountered in our targeting experiments. The second factor influencing the extended but unspecific tropism relates to the remarkable observation that as long as our selected vectors conferred any transgene expression in vivo, it invariably also occurred in the heart in addition to the target tissue. Heart expression of these vectors was even stronger than in wild-type AAV vectors. This is congruent with previous studies describing increased heart transduction upon modification of the VP3 region R484E/R588E In our studies, library-derived vector capsids  selected on primary murine breast cancer cells et al. recently described that peptides containing a net negative charge and inserted at the AAV capsid position 587 are prone to confer a heparin sulfate proteoglycan non-binding phenotype which correlates with liver and spleen de-targeting in mice Perabo in vivo screening of AAV-2 libraries allows for selection of vectors with an extended tropism towards the tissue of interest. Vectors targeting the endothelial cell layer in vivo might be used to deliver anti-angiogenic genes such as endostatin in order to block neovascularization and tumor growth in vivo and AAV de-targeting from the liver may further be optimized by using tumor specific expression systems such as the hTERT promoter Despite its obvious limitations, in vivo selections, especially if it has to be administered systemically. Furthermore, the helper-dependent selection requires a near to complete adenoviral infection of the target tissue in order to maximize the amount of clones that can be amplified. Using currently available helper viruses, this can not be achieved in all organs and tissues in vivo, particularly in light of the fact that many target tissues or cell types are not susceptible to adenoviral infection after systemic administration. In addition, it is not clear (and at least in the tumor tissue used in our study very questionable) if AAV can replicate in mouse tissue efficiently. Our novel selection method addresses these points. Relevant parts of the genomes of tissue-targeted library viruses are amplified via nested PCR. We distinguished between three alternative selection pathways, all of which are based on the amplification and enrichment of tissue-homed AAV library particles by PCR during the selection process. Pathway A is a cell-based in vitro selection approach in which genomes of internalized library viruses are amplified while non-internalized viral particles are eliminated. Although our PCR selection protocol does not exclusively force the selection for AAV capsids that mediate gene expression, we demonstrate the functionality of this technical approach in that clones sharing a common peptide motif (RGDXXXX) were recovered by screening on primary murine breast cancer cells and conferred efficient transduction of these cells, even in a cytotoxic suicide gene transfer approach. Similar peptide motifs have been selected on PC3 prostate carcinoma cells in vivo after systemic administration and has therefore been widely used to target cellular integrins expressed in the tumor tissue of xenograft mouse models V class of integrins. However, we consider this unlikely because the clones did not show preferential homing to tumor tissue in vivo and RGD as a tripeptide sequence by no means specifically binds to this class but also to other classes of integrins. In fact, 12 of the 20 currently known integrins recognize a certain RGD-containing sequence as their ligand In previous work on AAV libraries, internalized virus particles were amplified by adenoviral delivery of helper proteins in vivo. These pathways take into account several hurdles that limit viral receptor targeting under in vivo conditions. Thus, tissue homing particles with weak or unspecific binding capacities toward their targets are eliminated by host clearance mechanisms or by homing to other tissues, although the screening process does not guarantee that the selected clones confer gene expression in addition of homing to the target tissue. Upon using selection pathways A, B, or C for tumor targeting, the enriched peptide sequences varied depending on the respective selection pathway, indicating that the most suitable screening conditions may have to be evaluated for each individual target tissue.Pathways B and C aimed at selection of virus capsid variants after systemic administration of AAV libraries in vitro on various target cells, these results are a significant step forward and profoundly expand our knowledge on the mechanisms involved in the in vivo gene transduction of vectors derived from biopanning of AAV peptide libraries. In addition, they overcome some of the limitations observed in a recent report by Grimm et al.in vivo based on topical application to the airways (as opposed to systemic administration like in our study). Consequently, while two selected capsid mutants  in this study mediated gene transfer to certain cell types in the lung tissue after administration by inhalation, they failed to mediate vector retargeting after systemic administration. Interestingly, the NSSRDLG peptide insert described in this study was also described in previous studies on other cell types DLGSARA insert isolated from breast cancer tissue in the work presented here. Yet, both peptides probably bind to different receptors as the in vivo tropism seems to be very different which indicates that, at least for AAV, the whole peptide sequence rather than parts of a sequence motif has to be considered to evaluate its tropism.Compared to the previous work in which AAV libraries were selected et al.et al.in vivo selection.In addition, the two selections (in Grimm in vivo biopanning with a systemically administered random AAV peptide library over multiple selection rounds. We show that vectors displaying in vivo-selected peptides have a significantly improved transduction profile in breast cancer or lung tissues after systemic administration. These findings demonstrate the superiority of AAV clones selected in vivo over clones selected in vitro, as long as in vivo transduction is required. Unintended cardiac transduction by selected clones remains the major limitation to be addressed in subsequent studies, e.g., by further functional mapping of the capsid. Our findings broaden the understanding of AAV transduction profiles in vivo, the functionality of random AAV display peptide libraries and, even beyond the specific targets tumor and lung, are an important step in the development of targeted AAV gene vectors.This is the first report of


Dear Sir,-3We would like to report a case of a 38-year-old man who suffered second- and third-degree burns measuring approximately 3 × 12 cm to his right perianal region as the result of contact with a faulty car seat heater (Fig 1Car seat technology has added numerous complex features to modern cars, including seat heaters, seat coolers, and computerized controls to adjust seat position. Once considered a luxury item, electric heated car seats are now commonplace and have been known to malfunction and become dangerously hot, which may cause third-degree burns. The surface temperature at the heated car seat may reach 120°F. This temperature may cause localized deep and even life-threatening burns within 10 minutes.The seat heaters on every car should be thoroughly tested to increase their safety and have a maximum temperature setting.The car manufacturers should consider posting warnings in their owners' manuals highlighting the potential danger of burns due to heated seats.We hope that this case report will succeed in raising awareness of this problem.


It is understood that chronic allograft failure occurs as a result of alloimmune and non-alloimmune injury. Dendritic cells (DC) are thought to be crucial in regulating immune airway damage and interactions with epithelial cells are likely. Studies in human lung transplantation are limited, however, and the available literature on DC is inconsistent. This study focused on the ex vivo influence of primary bronchial epithelial cells derived from lung allografts on DC differentiation.Epithelial cell conditioned media (ECCM) were added to monocytes differentiating into DC under the influence of interleukin-4 and granulocyte macrophage-colony stimulating factor. The resultant cells were compared with DC cultured without ECCM and with monocyte-derived macrophages. Expression of typical DC  and macrophage  markers was assessed by flow cytometry. Phenotypical assessments were complemented by functional studies of mannose receptor-mediated phagocytosis (FITC-dextran uptake) and antigen-presenting capability (mixed lymphocyte reactions).Cells exposed to ECCM expressed significantly lower levels of CD1a than unexposed DC. CD14 expression and phagocytic function were increased. ECCM cultured cells also expressed lower levels of T cell co-stimulatory molecules, secreted an anti-inflammatory cytokine profile and had significantly reduced antigen-presenting capability.Using phenotypic and functional approaches, this study has shown that ECCM from lung allografts drives the production of macrophage-like cells from monocytes rather than DC. The data suggest that epithelial cells may restrain airway DC and potential alloimmunity. It is unclear whether the observed effect is specifically seen in lung transplant recipients or is a general property of bronchial epithelial cells. This may reflect a homeostatic inter-relationship between airway epithelial and DC populations relevant both to lung allografts and the lung more generally. Lung transplantation is an accepted treatment for end stage lung disease in carefully selected patients, but success is compromised by chronic allograft dysfunction.et al showed increased numbers of DC in chronically rejecting lung allografts,et al indicated that DC co-expressing the co-stimulatory molecules CD80 and CD86 were more frequent in allografts with chronic rejection.5Yousem et alThe airway epithelium is a recognised focus of damage in chronic allograft dysfunction, but it is also increasingly accepted that airway epithelial cells may play a key part as effector cells through the production of a wide range of inflammatory and immunomodulatory cytokines and growth factors.To our knowledge, there are no studies in primary epithelial cell cultures from lung allografts evaluating the potential for epithelial cells to modulate the production of dendritic cells from monocytes. In this study, recognising the lack of specificity of phenotypic markers alone, we have investigated phenotypic and functional properties for DC using a previously validated ex vivo DC culture system. Using a coordinated and multi-parametric approach, we tested the hypothesis that epithelial cell conditioned media can modulate phenotypic and functional properties of DC.Clinically stable lung allograft recipients free from chronic allograft dysfunction were used to obtain primary epithelial cells. Clinical details are shown in Our post-transplant patients undergo surveillance bronchoscopy at 1 week, 1, 3, 6 and 12 months as well as further bronchoscopy if indicated on clinical grounds. All patients underwent pulmonary function testing and an assessment of clinical status was made based on standardised criteria.11Bronchoalveolar lavage (BAL) fluid was obtained from either the lingula or right middle lobe and sent for routine microbiological testing. Bronchial brushings (n = 4–6) were obtained from subsegmental bronchi using a standard single-sheathed nylon cytology brush  and dispersed in 5 ml phosphate buffered saline . Transbronchial specimens were taken from either the right or left lower lobe and sent for histopathological examination to exclude acute vascular rejection based on standard ISHLT criteria.12Primary bronchial epithelial cells were obtained from bronchial brushings as previously described.6 cells/ml for 6 days in 0.5 ml BEBM and 0.5 ml RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. To generate DC, IL4 and granulocyte-macrophage colony stimulating factor (GM-CSF)  and to generate macrophages (Mph), macrophage colony stimulating factor (M-CSF)  were added to the monocyte cultures on days 0 and 3. After 6 days, cells were stimulated with lipopolysaccharide  or were left untreated. After 24 h the supernatants were harvested and assayed for IL12p70 and IL10 by ELISA, and the DC and MPh were extensively washed before performing functional assays and flow cytometry.Monocytes were isolated from freshly drawn peripheral blood from healthy donors or buffy coats, as described previously.Monocytes were cultured under DC-generating conditions as described above, but with 0.5 ml ECCM instead of BEBM. The ECCM-modified DC cultures are referred to as antigen-presenting cells (APC) throughout the paper.The following antibodies were used for cell surface marker analysis: CD1a , CD14 (M5E2), CD68 (27–35), CD86 (2331 FUN-1), CD83 (Hb15e) and HLA-DR . Isotype-matched control antibodies were used to confirm that staining was specific (data not shown). Cells were centrifuged and resuspended in FACS buffer . Human IgG  was added with antibodies to prevent Fc receptor binding. To detect intracellular CD68, a permeabilisation step was included . Cells were incubated on ice for 30 min, washed and resuspended in FACS buffer. Data were collected on a Becton Dickinson FACScan and analysed using FlowJo .Fluorescent-labelled dextran  was used to assess the mannose receptor (MR)-mediated phagocytic capacity of the cell types. Cells were incubated with FITC-dextran at 37°C for 1 h while a control group was left on ice to exclude extracellular binding of FITC-dextran. Cells were extensively washed and intracellular FITC-dextran was quantified by flow cytometry.4 DC, APC or MPh were cultured with 1×105 allogeneic CD3+ T lymphocytes in 200 μl cultures in a 96-well plate. Supernatants were harvested after 6 days and assayed for interferon γ (IFNγ) by ELISA. Proliferation was assessed by incorporation of 3H-thymidine for the last 8 h of culture by scintillation counting .1×10Cytokine levels in the supernatants were measured by specific sandwich ELISA performed with commercially available matched antibody pairs. The following cytokines were measured: IL8 and IL15 (R&D Systems), IFNγ, IL6, IL10 and IL12p70 (BD Pharmingen).t test for the following comparator group pairs: DC and APC; APC and MPh. Only significant results (p<0.05) are indicated in the figures.Statistical software used for the analysis was GraphPad Prism Version 4. In the upper panels of Phenotypic markers assessed were the DC marker CD1a, the MPh markers CD14 and CD68, the antigen presenting molecule HLA-DR, the classical DC maturation marker CD83 and the T cell co-stimulatory molecule CD86. The salient findings of these experiments showed that ECCM drove the production of MPh-like cells from monocytes. In the APC population there were significantly fewer cells expressing the DC marker CD1a than in the DC population, contrasting with significantly more cells expressing the MPh marker CD14. APC also expressed higher levels of CD68, but this was not statistically significant.Introduction of LPS stimulation to the protocol led to a significant upregulation of HLA-DR; CD83 and CD86 on both DC and APC, but not MPh. However, the levels of CD83 and CD86 were significantly lower on LPS-activated APC than on LPS-activated DC, while expression levels of HLA-DR were similar.We also measured the production of the anti- and pro-inflammatory cytokines IL10 and IL12p70, respectively, by DC, APC and MPh that had been activated by LPS for 24 h . APC proBecause the phenotype of APC resembled the phenotype of MPh rather than DC, we hypothesised that the functional characteristics of APC would also be more macrophage-like. To test this hypothesis we assessed functional abilities that are typical for either MPh (phagocytosis) or DC (T cell stimulation).Together these data indicate that ECCM skew monocyte-derived DC towards a macrophage-like population in terms of phenotype and function.This study has shown that conditioned media from lung allograft primary bronchial epithelial cells drives the production of macrophage-like cells from monocytes rather than DC. Although there was biological variability, a broad range of phenotypic and functional markers consistently supported this finding. Our data suggest that epithelial cells from allograft recipients promote production of MPh while restraining the airway DC population and potential alloimmunity. It is unclear whether the observed effect is specifically seen in lung transplant recipients or might be a general property of bronchial epithelial cells. It remains to be studied whether this is relevant both to lung allografts and to the lung more generally.The biology of professional APCs in the airway is likely to be of key importance in the alloimmune component of chronic allograft dysfunction which is commonly referred to as chronic rejection. There are few studies in lung transplantation, however, and these are restricted to phenotypic approaches using different markers which report conflicting data. Two studies indicate that, in chronic rejection, there are increased numbers of DC5It is accepted that the local environment to which DC are exposed is a key factor in their maturation and function.et al using lung A549 and airway BEAS-2B epithelial cell lines.The potential for epithelial cells to interact with DC was recently studied by Regamey et al. Using accepted markers for DC and macrophages we consistently showed that conditioned media from allograft recipient primary epithelial cells favoured the production of macrophage-like cells from monocytes. Phenotypic markers were corroborated by functional phagocytic and immunostimulatory assays. Our data showed that incubation of monocytes with patient-derived ECCM consistently yielded a population of cells with low expression of DC markers and poor T cell stimulatory ability. In contrast, we observed significant expression of macrophage markers and competent phagocyte function. The conditioned media from allograft primary epithelial cells did not contain detectable levels of IL15 as measured by ELISA (data not shown), which may reflect differences between primary epithelial cells and cell lines and/or differences in the experimental set-up .Our findings are not consistent with those of Regamey et al was that we used primary epithelial cells sourced from lung transplant recipients. It is of interest that a previous study by Ohtoshi et al using upper airway epithelial cells from polyps drove the production of MPh from monocytes, although this was in atopic but otherwise healthy volunteers and not a transplant-related study investigating DC markers.19A main difference between our work and the study by Regamey Danger signals provided by stimuli such as infection in the allograft may be important in promoting alloimmunity.21In the present study we showed that monocytes incubated with ECCM and then stimulated with the Toll like receptor 4 agonist LPS upregulated HLA-DR, CD83 and CD86, which are markers of DC activation. However, the T cell stimulatory capacity of ECCM-induced APC was not enhanced. It is likely that the high production of the anti-inflammatory cytokine IL10 by LPS-activated APC will have counteracted the T cell co-stimulatory activity of CD83 and CD86, but this requires further investigation. Interestingly, the ECCM-induced APC resemble regulatory DC  described previously by us and others,Our data emphasise the need for primary cell data to be assessed along with experiments involving cell lines, although primary studies are themselves limited by technical difficulty and the fact that they are resource intensive. Further studies would be useful to investigate conditioned media sourced from epithelial cells from subjects with chronic allograft dysfunction. This was beyond the scope of this present study, since our experience is that such cultures commonly fail due to antibiotic-resistant patient-derived infection.Our experiments were all performed using epithelial cells at early passage (I–II) in cell cultures free from transplant maintenance therapies. We do not expect previous patient immunosuppression to be responsible for our findings, but transplant medications might affect the interplay between epithelial cells and DC in the airway. This could also be an avenue for further research. An aim of this could be to elucidate the most appropriate balance between appropriate immunosuppression and preservation of airway homeostasis, with maintenance of epithelial function and appropriate networking with airway DC representing a desirable goal.