Furthermore, we show that Cxcl10 upregulation in MECs is promoted by interferon-λ and that Usp18 is a novel inhibitor of interferon-λ signalling. Knockdown of the interferon-λ specific receptor subunit IL-28R1 in Usp18 deficient MECs dramatically enhances tumour growth. Taken together, our data suggest that targeting Usp18 may be a viable approach to boost antitumour immunity while suppressing the protumour activity of the immune system.The theory of cancer immunoediting refers to mechanisms by which the immune system can suppress or promote tumour progression. A major challenge for the development of novel cancer immunotherapies is to find ways to exploit the immune system's antitumour activity while concomitantly reducing its protumour activity. Using the PyVmT model of mammary tumourigenesis, we show that lack of the Usp18 gene significantly inhibits tumour growth by creating a tumour-suppressive microenvironment. Generation of this antitumour environment is driven by elevated secretion of the potent T-cell chemoattractant Cxcl10 by Usp18 deficient mammary epithelial cells (MECs), which leads to recruitment of Th1 subtype CD4 Novel effective treatments for cancer remain in high demand and targeting so called immunomodulators to enhance antitumour immunity has garnered considerable interest. Particular cells of the innate and adaptive immune system can, in conjunction with secreted molecules, function as potent extrinsic suppressors of tumourigenesis and cancer progression . A number of reports using transgenic animal models showed that mice deficient in IFNs or their receptors are more susceptible to spontaneous or drug-induced cancer  mouse model for breast cancer  lines derived from PyVmT/Usp18 KO tumours. These cell lines were transduced with either empty vector retrovirus (KO) or with Usp18 expression retrovirus (KO + Usp18). Levels of proliferation marker Ki67 were mostly unchanged in tumour tissues of PyVmT/Usp18 KO deficient mice . In concficiency  suggestificiency , nor theficiency  was signficiency . Interesficiency  that coun assays .+ T cells in tumours of PyVmT/Usp18 KO mice compared to PyVmT/Usp18 WT mice  cells and F4/80+macrophages in PyVmT/Usp18 KO tumours though the difference did not reach statistical significance. Tumour associated myeloid derived suppressor cells (CD11b+/Gr-1+), however, were not changed. We further confirmed an increase of CD4+ T cells in mammary tumours of Usp18 KO mice by immunofluorescence studies  stained sections of mammary tumours from 13-week-old mice revealed a reduction in tumour progression in PyVmT/Usp18 KO mice. We distinguished early and late carcinoma from adenomas based on Lin et al's recommendations for the classification of mouse mammary tumour pathology  cells  and CD4+ T cells present in the tumour  inducible genes, they are also induced by type I IFN and TNF-α but preferences and strength of induction vary between the three  and plasmacytoid DCs (pDCs) being the most important sources . pMSCV-puro-hUSP18 and pCDNA3.1-USP18 were previously described  and frozen in liquid nitrogen. For histochemical analysis paraffin sections were stained with H&E according to standard protocols. Immunostainings were performed on paraffin or frozen sections using VECTASTAIN ABC peroxidase/DAB staining kits (Vector Laboratories). For immunofluorescent staining, frozen sections were prepared at 8 µm thickness and stained with antibodies against CD4 followed by FITC conjugated secondary antibodies. Cell nuclei were counterstained by 49,69-diamino-2-phenylindole (DAPI). Images were captured using an Olympus camera model DP71 on a Olympus BX51 microscope at 100× or 200× magnification.PROBLEM:While the understanding of the role of the immune system in cancer progression has made significant progress over the past decades, the successes of cancer immunotherapies have been inconsistent. For instance, in the case of anticancer vaccines only a minority of patients actually benefit from vaccine-induced T cells, which is likely due to inefficient homing of tumour-specific T cells to the tumour site. We hypothesize that new therapies that can efficiently attract tumour-suppressive immune cells to the tumour area will improve efficacy of current cancer therapies.RESULTS:We report that Usp18 deficient MECs secrete elevated levels of the T-cell chemoattractant Cxcl10. Accumulation of this chemokine leads to enhanced recruitment of Th1 cells and generation of a tumour-suppressive cytokine environment resulting in inhibition of mammary tumour growth. Furthermore, we show that Usp18 is a novel negative regulator of IFN-λ signalling, which promotes upregulation of Cxcl10 in MECs lacking Usp18.IMPACT:We identified Usp18 as a regulator of the tumour microenvironment and promising candidate for breast cancer immunotherapy. Since IFN-λ acts exclusively on epithelial cells, a combined treatment with a Usp18 inhibitor will locally boost the antitumour immune response with minimal off-target effects. This combination of a Usp18 specific inhibitor with IFN-λ treatment may be of great benefit for the treatment of epithelial cancers.Protein extracts were prepared from mammary tumours, isolated tumour cells or established cell lines by lysis in modified RIPA buffer as described previously .Single cell suspensions were established from spontaneous mammary tumours arising in FVB MMTV-PyVmT females. Tumours were minced and incubated at 37°C for 3 h in 5 ml of Ham's F12K medium containing 1 mg/ml collagenase (Roche), 2 mg/ml soybean trypsin inhibitor (Sigma) and 2% BSA. After addition of FCS-containing medium, the suspension was passed through a 70 µm nylon filter (Fisher Scientific) and red blood cells removed with ACK lysing buffer. Single cells were pelleted by centrifugation and resuspended in PBS for immediate flow cytometric analysis. Stained cells were analyzed by a FACSCanto cytometer (BD Biosciences). For establishment of MEC cell lines cells were cultured in DMEM:F12 (1:1) medium mix containing 5% FCS, 2.5 µg/ml amphotericin B, 10 µg/ml penicillin–streptomycin and MITO+ (BD Biosciences). Non-epithelial cells were removed by differential passaging and epithelial origin of established cell lines confirmed by CD326 staining after five passages (CD326In vitro cultured MECs (1 × 106) resuspended in 1 mg/ml matrigel solution were injected into the abdominal mammary fat pads of female mice. Growing tumours were monitored once a week with a calliper.3) were seeded in each well of a 96-well plate on day 0. Cell growth was monitored for 1 week with CellTiter 96® AQueous One Solution Reagent according to manufacturer's instructions (Promega).Cells . The cells were suspended in serum-free DMEM and then added to the upper chamber at a density of 3 × 10Total RNA from tumour tissue or MECs was isolated using RNeasy (Qiagen) according to the manufacturer's instructions. For qRT-PCR analyses, equal amounts of RNA were reverse-transcribed by qScript (Quanta Biosciences) and the resulting cDNA templates were subjected to qRT-PCR using SYBR Green detection system on the CFX96 thermal cycler (BIO-RAD). Primer sequences are available upon request.T-test, using p < 0.05 as indicative of statistical significance. Kaplan–Meier tumour-free survival data were compared using the log-rank test.Statistical significance was evaluated by a paired


The authors wish to add the following to their Funding section: "This initiative is funded by the CTSI (grant by number 1 UL1 RR029893)."


We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart.Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy.Derivatisation of a stabilised DOTA-BN(7–14)[Cha2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios . In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour growth. The therapeutic efficacy was highest for the PEGylated derivative of high specific activity administered in two fractions (2 × 20 MBq = 40 MBq) at day 0 and day 7 .The specificity and the binding affinity of the BN analogue for BN177Lu-labelled BN-based radiopharmaceutical and provide a protocol for targeted radionuclide therapy with a beneficial anti-tumour effectiveness and a favourable risk-profile at the same time.PEGylation and increasing the specific activity enhance the pharmacokinetic properties of a  Using BN conjugates for specific delivery of radionuclides into the above-mentioned tumours is therefore a promising strategy for diagnostic and therapeutic purposes.Prostate and breast cancers are the most frequently diagnosed forms of cancer in the USA. Especially in addressing metastatic and small-volume diseases, it is essential to investigate, alongside conventional therapies, alternative treatments, such as peptide receptor radionuclide therapy (PRRT). The fact that certain tumour types overexpress, receptors for peptide-hormones provide the basis for successful use of radiolabelled peptide analogues as tumour tracers in nuclear medicine. The mammalian gastrin-releasing peptide receptor BN2/GRP),2 is par/GRP,2 i2/GRP)-5. The tin vivo, which might prevent sufficient localisation at the target site. Furthermore, high accumulation and retention in healthy organs, which express the BN2/GRP receptor, increase the risk of side effects. Moreover, kidney toxicity, which was observed and investigated in PRRT with somatostatin analogues in clinical studies[BN analogues, however, present certain problems regarding therapy. They show poor enzymatic stability  studies-8, may a2/GRP receptors. Different BN analogues were labelled with diagnostic single-photon emission computed tomography (SPECT) and positron emission tomography (PET) radionuclides, such as 111In[99mTc[18 F[68 Ga[64Cu[2/GRP receptor-positive lesions. However, only a few radiolabelled BN analogues have been tested in clinics on their diagnostic potential[2 and DOTA-PESIN, have been rated in preclinical investigations on their potential for radionuclide therapy[177Lu  or with 213Bi . The in vitro and in vivo evaluations of our 177Lu-DOTA-Lys-BN analogue  showed pharmacokinetic properties which are comparable to that reported for the above-mentioned BN analogues. Therefore, we wanted to improve the radiotherapy-relevant characteristics further by PEGylating 177Lu-DOTA-Lys-BN.Until now, the research has focused on optimising BN conjugates for nuclear imaging of cancer which overexpresses BNas 111In-11, 99mTn[99mTc-15, 18 FTc[18 F-18, 68 GF[68 Ga,20 and 6Ga[64Cu, and werotential. So far, therapy-25. They99mTc(CO)3-labelled PEGylated BN analogues showed that PEGylation is an effective strategy to improve the therapy-relevant characteristics, which include higher tumour uptake, improved tumour retention and lower uptake into non-target tissue. The PEG entity of 5 kDa was established as the optimal PEG size because it improved these features best[Our preclinical study with a series of res best.7-Trp8-Ala9-Val10-Gly11-His12-Cha13-Nle14-NH2) containing a β3hLys-βAla-βAla spacer  was performed by PEGylation of the ϵ-amino group of the lysine residue. The resulting PEGylated BN . All analyses were 2-tailed and considered as type 3 ; P < 0.05 was considered statistically significant.All data are presented as mean ± SD. The 177LuCl3 (714.3 GBq/μmol) were added to a mixture of 20 μl ammonium acetate solution , 84 μl HCl (0.04 M), 5 μl ascorbic acid solution (0.05 M) and 5.6 nmol of BN analogue . The final solution (pH 4.5) was heated at 75 °C for 15 min , unlabelled BN analogue was added to the high specific labelling solution to reach the respective concentration.For high specific activity labelling, 16 μl of approximately 400 MBq The labelled analogues were incubated with human plasma  at 37 °C for various time intervals up to 12 days. After incubation, proteins were precipitated with acetonitrile/ethanol (1:1) and TFA (0.1%) and then centrifuged. The supernatant was analysed with RP-high-performance liquid chromatography (HPLC) equipped with a radioactivity detector. The radioactivity chromatograms showed different peaks which corresponded to the intact peptide and the different degradation products. The experiments were performed two times.For internalisation, PC-3 cells at confluence were placed in six-well plates and left to attach overnight. Cells were incubated with the labelled analogues (4 kBq) in culture medium for 0.5, 1, 2, 4 and 24 h at 37 °C. Non-specific binding was determined with 1 μM unlabelled BN(1–14). After the different incubation times, cells were twice washed with cold phosphate buffered saline (PBS) to discard unbound peptide. Surface-bound activity was removed by two 5-min acid washes . Afterwards, the cells were washed with cold PBS, and lysed with 1 N NaOH twice. Surface-bound and internalised radioactivities were measured in the gamma counter.For externalisation, PC-3 cells were incubated with the labelled analogues (60 kBq) in culture medium at 37 °C for 1 h. After incubation, the supernatant was discarded, and the cells were twice washed with cold PBS. The cells were then incubated again at 37 °C in culture medium for 0.5, 1, 2.5, 5 and 24 h. At each time point, the supernatant was collected, the cells twice washed with cold PBS and lysed with 1 N NaOH. The supernatant (released radioactivity) and the cells  were measured in the gamma counter. All experiments were carried out two to three times in triplicate.6 PC-3 cells in 150 μl culture medium without supplements. The tumours were allowed to grow for at least 3 weeks. On the day of the experiment, the mice (3 to 6 per group) received the radioactive conjugates intravenously. For the biodistribution studies, the mice were injected with different specific activities of the radiolabelled BN analogues . Receptor-blocking studies were performed using 100 μg of unlabelled BN(1–14) co-injected with the corresponding radiolabelled BN analogue. At 1, 4 and 24 h post injection (p.i.), the animals were euthanised and dissected. Blood, tumours and various healthy tissues and organs were collected, weighed and examined for radioactivity. Results are expressed as percentage of injected dose per gram of tissue (% ID/g).All animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal experimentation established by the Swiss Academy of Natural Sciences. Biodistribution studies were performed with 6- to 8-week-old female CD-1 nu/nu mice (20 to 25 g) purchased from Charles River Laboratories . For the induction of tumour xenografts, each mouse received subcutaneously 8 × 10n = 3 per group). Under the assumption of rapid accumulation (uptake at 0 h p.i. corresponds to the uptake at 1 h p.i.), the cumulative radioactivity in each tissue was calculated (MBq/h) taking biologic elimination and physical decay into account up to 24 h p.i. and afterwards only physical decay up to 400 h p.i. The absorbed tumour doses of the mouse experiments were extrapolated from the sphere model doses which were calculated by using the software OLINDA . The S values for all other tissues of mice were taken from E Larsson[S value (mGy/(MBq☆s)). The dose (in Gy) was calculated by multiplying the absorbed dose (mGy/MBq) with the amount of radioactivity injected (20 MBq). The AUC-estimate for an adult male was obtained by multiplying the AUC of the mice (MBq/h) with a factor consisting of  × organ weightadult male. The subsequent dose calculation was performed using the adult male model of the software OLINDA.The absorbed doses to PC-3 tumours and critical organs were calculated from the biodistribution studies , the tumours had an average volume of 85 mm3. The animals were divided into six groups of six mice (Table5k-Lys-BN at a peptide amount of 3.0 nmol (group B). The treated mice received two equal doses of 20 MBq i.v. either of 177Lu-DOTA-PEG5k-Lys-BN (groups C to E) or of 177Lu-DOTA-Lys-BN (group F) at a peptide amount of 0.3 or 3.0 nmol. The injections were performed either at days 0 and 14 or at days 0 and 7 , and V0 was the measurement at day 0. If a tumour disappeared, Vt was set to 0. Mice were removed from the study promptly upon fulfilling one or both of the following criteria: a tumour volume of  ≥ 1.5 cm2 or total body weight loss of ≥ 15%. Upon euthanasia, tumours were collected and embedded in TissueTek  and frozen for autoradiography.Therapy studies were conducted in mice bearing PC-3 xenografts. The tumour was assumed to be an ellipsoid, and its volume was calculated with the formulace Table. The con99mTc-DMSA tests. An untreated control group of mice (group G), a treated group receiving two doses of the 177Lu-DOTA-PEG5k-Lys-BN analogue at high specific activity (group H) and a treated group of mice getting two doses of the 177Lu-DOTA-PEG5k-Lys-BN analogue with low specific activity (group I). 99mTc-DMSA scans with SPECT/computed tomography (CT) were obtained 43, 71 and 111 days after therapy, 2 h after i.v. injection of about 30 MBq 99mTc-DMSA. SPECT scans were acquired with anaesthetised mice during 20 min using 15 projections/min. The images were obtained on an X-SPECT-system  equipped with a single head SPECT device and a CT device. SPECT data were acquired and reconstructed with LumaGEM . CT data were acquired with an X-ray CT-system (Gamma Medica) and reconstructed with the software CoBRA . SPECT and CT data were combined with the software IDL Virtual Machine . The images were generated with Amira (version 4.0). Quantification of the amount of radioactivity in a volume of interest over the kidneys was performed with Amira . Detected counts in the volume of interest were normalised to 1 MBq ID.Three groups of four mice each . The 177Lu-DOTA-Lys-BN was rapidly degraded by proteases in human plasma. After 5 days of incubation, it was almost entirely metabolised, and only 13.8 ± 5.7% remained intact. PEGylation resulted in a marked increase in protease stability; 51.8 ± 0.9% of 177Lu-DOTA-PEG5k-Lys-BN still remained intact after 5 days of incubation and 43.7 ± 0.5% after 11 days. Moreover, the half-life of 177Lu-DOTA-Lys-BN in human plasma was 1.2 ± 0.3 days, whereas the half-life of 177Lu-DOTA-PEG5k-Lys-BN was 6.7 ± 1.4 days . The PEGylated analogue showed a significantly lower and slower internalisation into PC-3 cells. After incubation for 4 h, the internalised fraction was 3.3 ± 1.2%. Externalisation studies revealed 63.1 ± 4.0% of the internalised 177Lu-DOTA-Lys-BN externalised within the first 2.5 h. After 24 h, only 13.5 ± 7.2% of the internalised fraction was found in the cells. In contrast, the externalisation of the PEGylated analogue was slower (24.2 ± 1.3% after 24 h).in vivo in mice with PC-3 tumour xenografts performed with BN conjugates at an injected peptide amount of 0.075 nmol (Table177Lu-DOTA-Lys-BN (1.88% ID/g) and 177Lu-DOTA-PEG5k-Lys-BN (3.43% ID/g) was found 1 h p.i., the latter being significantly higher. Thus, the enhanced enzymatic stability induced by PEGylation compensated for the lower receptor affinity of DOTA-PEG5k-Lys-BN. Furthermore, the tumour washout was slightly slower for the PEGylated analogue. Thus, 1.04% ID/g was found for the 177Lu-DOTA-PEG5k-Lys-BN 24 h p.i., whereas only 0.54% ID/g of 177Lu-DOTA-Lys-BN remained in the tumour. At 1 h p.i., both analogues showed their highest uptake in the pancreas, which expresses GRP receptors . The liver uptake of both 177Lu-DOTA-Lys-BN and 177Lu-DOTA-PEG5k-Lys-BN was low . Kidney accumulation was higher for the 177Lu-DOTA-PEG5k-Lys-BN in comparison with that of the non-PEGylated analogue at 1 h p.i. . The renal clearance, however, was fast for both analogues . The conjugation of a PEG5k entity resulted in a significantly longer blood circulation during 1 h p.i., but both derivatives were completely cleared from blood within 24 h p.i. (TableThe effect of PEGylation was tested ol Table. The higSpecificity for GRP receptors could be demonstrated by a co-administration of non-radioactive BN(1–14). Thus, only the uptake in the receptor-expressing tissues such as the pancreas, colon and tumour was markedly reduced (>70%), whereas the inhibition was slightly less effective for the PEGylated BN analogue Table.177Lu-DOTA-PEG5k-Lys-BN, however, resulted in lower tumour-to-blood ratios at 1 and 4 h p.i. compared with that of the non-PEGylated compound. 177Lu-DOTA-PEG5k-Lys-BN showed a twofold increase in the tumour-to-pancreas ratio at all time points and in tumour-to-kidney and tumour-to-liver ratios at 24 h p.i.  were administered. This showed that the uptake in the receptor-expressing tissues such as the pancreas and colon was markedly reduced by applying a high amount of PEGylated peptide (3.0 nmol). The tumour uptake was also reduced by 29% and 42% at 1 h p.i. and 24 h p.i. after injection of a high amount of peptide . The only ratios which revealed a higher value with a high amount of peptide (3.0 nmol) were the tumour-to-pancreas ratios  in a single dose, the absorbed doses were calculated to be 0.36 Gy/MBq for the murine kidney, 0.002 Gy/MBq for the blood, 0.02 Gy/MBq for the pancreas and 0.19 Gy/MBq for the tumour. After the application of 177Lu-DOTA-PEG5k-Lys-BN at a high amount of peptide (3.0 nmol), however, the absorbed doses to the murine kidney, blood, pancreas and tumour were calculated to be 0.50, 0.002, 0.006 and 0.11 Gy/MBq, respectively. The estimate for an adult male resulted in absorbed doses to the kidney, blood and pancreas of 9.4, 0.4 and 21.3 Gy/GBq (0.3 nmol) and 12.3, 0.4 and 5.2 Gy/GBq (3.0 nmol), respectively.After applying n = 6) and three additional groups of mice without tumours . All groups of mice which received the 177Lu-labelled BN analogues (C to F) clearly showed a reduction of tumour growth in comparison with groups A and B which received only PBS or unlabelled BN analogue. The treatment with the non-PEGylated 177Lu-labelled BN analogue  significantly decreased the PC-3 tumour growth rate with respect to that of group A and exhibited an inhibition of 53% 3 weeks after the first dose . However, the 177Lu-DOTA-PEG5k-Lys-BN analogue of low specific activity (group C) exhibited only an inhibition of 36%. The most effective tumour growth inhibition of 73% (3 weeks after the first dose) was observed when the second dose of the PEGylated BN analogue of high specific activity was applied 7 days after the first dose  receiving the radiotracer of high specific activity was 76,397 counts/kidney, whereas the uptake of the treated animals receiving the radiotracer of low specific activity (group I) was 74,949 counts/kidney. Seventy-one days after therapy, there was no significant difference in the renal 99mTc-DMSA uptake between groups G, H and I ; 111 days after therapy, there was also no significant difference in the renal 99mTc-DMSA uptake between these three groups of mice.Forty-three days after therapy, the renal 2, AMBA  and DOTA-PESIN , have been evaluated for PRRT[177Lu or 213Bi and showed anti-tumour effectiveness in mice with PC-3 xenografts. Regarding in vitro evaluation and biodistribution data, our 177Lu-DOTA-Lys-BN analogue showed pharmacokinetic properties which are comparable to those of the above-mentioned BN analogues, except for the higher tumour uptake and the better retention profile of AMBA and DOTA-PESIN. Therefore, we wanted to improve the radiotherapy relevant characteristics further by PEGylating 177Lu-DOTA-Lys-BN.So far, only three optimised BN analogues, DOTA-8-AOC-BN(7–14)NHfor PRRT-25. ThesIn vitro, time-dependent cell uptake and internalisation showed slower binding kinetics for the PEGylated BN analogue. These findings are in line with the results of PEGylating other biomolecules reported in the literature[177Lu-DOTA-PEG5k-Lys-BN and explain why binding affinity of this analogue in vitro was slightly reduced  of 177Lu-DOTA-PEG5k-Lys-BN in human plasma was 5.6-fold higher in comparison with that of non-PEGylated 177Lu-DOTA-Lys-BN. In comparison to 177Lu-AMBA, which is more stable in human plasma (t1/2 = 38.8 h)[177Lu-DOTA-PESIN (t1/2 = 8.4 h)[in vitro half-life of 177Lu-DOTA-Lys-BN in human plasma (t1/2 = 28.8 h) was in the same range but was markedly higher with the PEGylated BN analogue (t1/2 = 160.8 h).Previously, we could confirm that PEGylation improves the stability of BN toward enzymatic degradation. In the o Figure. The hal 38.8 h) than 177= 8.4 h), the in 177Lu-AMBA and 177Lu-DOTA-8-AOC-BN(7–14) (HPLC purified) was injected per mouse[177Lu-DOTA-PESIN (0.2 nmol peptide)[177Lu-labelled BN analogues were injected. This 0.075 nmol is the nearest possible approximation to the 0.002 nmol without HPLC purification, which is desired in clinics. In comparison with 177Lu-AMBA, our 177Lu-DOTA-Lys-BN showed an approximately fourfold lower kidney uptake 1 h p.i., whereas the kidney uptake of the 177Lu-DOTA-PEG5k-Lys-BN analogue was 2.3-fold lower at 1 h p.i. Both compounds showed a faster clearance from the kidneys within 24 h p.i. Kidney accumulation and washout of our 177Lu-DOTA-Lys-BN and 177Lu-DOTA-PEG5k-Lys-BN were comparable to those of 177Lu-DOTA-PESIN (3.8 ± 0.34%ID/g at 1 h p.i.), even though Gelofusine and polyglutamic acid were co-administered with 177Lu-DOTA-PESIN for the reduction of renal uptake[177Lu-DOTA-Lys-BN and 177Lu-DOTA-PEG5k-Lys-BN at 1 and 24 h p.i. compared with that in 177Lu-AMBA  and 177Lu-DOTA-8-AOC-BN(7–14) [The biodistribution data, in which 0.002 nmol of er mouse, and thepeptide) were coml uptake. Furtherctively). However177Lu-DOTA-PEG5k-Lys-BN showed significantly higher tumour uptake at 1 h p.i. in comparison with the non-PEGylated counterpart. The higher enzymatic stability as well as the longer blood circulation may have compensated for the slower binding kinetics and the lower receptor affinity of DOTA-PEG5k-Lys-BN. In order to compare the cumulative radioactivity over 24 h of each conjugate in the tumour, the AUC value of 177Lu-DOTA-Lys-BN was arbitrarily set to 1. The comparison showed a relative AUC value of 1.6 (P < 0.0006) for 177Lu-DOTA-PEG5k-Lys-BN.177Lu-DOTA-PEG5k-Lys-BN retained in the tumour between 0 and 24 h p.i. The extended tumour retention for the 177Lu-DOTA-PEG5k-Lys-BN might be explained by the improved enzymatic stability of the peptide derivative, and the extended retention might be due to the enhanced permeation and retention in the tumour. On the basis of the biodistribution data with 177Lu-AMBA[177Lu-DOTA-PESIN[177Lu-labelled BN analogues. However, compared to 177Lu-DOTA-8-AOC-BN(7–14)[177Lu-DOTA-Lys-BN analogue showed a similar tumour uptake, but the uptake of 177Lu-DOTA-PEG5k-Lys-BN was higher. This comparison, however, must be looked at with due care because the study designs differ insofar as different peptide amounts were injected.The second hypothesis that PEGylation prolongs the tumour retention was also proven. Even though PEGylation lowered the tumour washout only slightly between 1 and 24 h p.i., there was more 7Lu-AMBA and 177LTA-PESIN, both BNBN(7–14)  affected the uptake into receptor-expressing tissues. The amount of 0.3 nmol was selected to approximate the 0.22 nmol of the AMBA therapy study because these amounts of 0.3 nmol have proven to be the limit for high specific labelling, i.e. the labelling is reproducible without any loss in yield. The amount of 3.0 nmol however was selected because a preliminary study (data not presented) had suggested that peptide amounts in this range markedly reduce the uptake into non-target receptor positive tissues. In comparison with a low peptide amount, applying a high peptide amount resulted in a marked reduction in pancreas and colon uptake which would lower the risk of radiotoxic side effects induced by radionuclide therapy  compared with 177Lu-DOTA-Lys-BN (group F) . The lower tumour accumulation of 177Lu-DOTA-PEG5k-Lys-BN of low specific activity resulted in a proportionally faster tumour growth. We could demonstrate that the reduced efficacy is not caused by the tumour growth-promoting effect of the higher peptide amount since unlabelled DOTA-PEG5k-Lys-BN (group B) did not induce tumour growth compared with the control group , resulted in a markedly higher therapeutic efficiency when Furthermore, it can be assumed that an increase in specific activity would achieve at least the same therapeutic efficiency as low specific activity, but the dosage injected would be lower.in vitro autoradiography  was applied 7 days after the first dose instead of 14 days (73% vs. 63% inhibition at day 21) (group D).Preliminary therapy studies [177Lu-DOTA-8-AOC-BN(7–14) (approximately 73% vs. approximately 79%) 3 weeks after the first dose. However, such a comparison is not fully conclusive since these therapy studies differ in tumour size at the beginning of therapy, injected peptide amount, administered radiation dose and injection interval.Comparing our study with the therapy studies with 7Lu-AMBA, 177Lu-DTA-PESIN (2 × 28 BN(7–14), we foun177Lu-DOTA-PEG5k-Lys-BN analogue were applied. This estimate implies that an administration of approximately 2 GBq of either low or high specific 177Lu-DOTA-PEG5k-Lys-BN analogue would result in absorbed kidney doses of approximately 18.8 or 24.6 Gy, respectively. These doses would not exceed the acceptable safe limit of 23 to 27 Gy[99mTc-DMSA scintigraphy which showed that there was no kidney damage in the mice treated with high or low specific 177Lu-DOTA-PEG5k-Lys-BN analogue (group H and I) since there was no significant difference in renal 99mTc-DMSA uptake of control and treated mice. Besides, serum analysis confirmed the absence of renal toxicity  and serum analyses, results of the preliminary therapy study and the results of the in vitro autoradiography of tumour sections are presented in the Additional file.Synthetic details of the PEGylation of the DOTA-Lys-BN analogue, experimental details of the octanol/PBS partition coefficient (log D) determination, details of the apparent receptor affinity (IC References[ferences,25,37-39Click here for file


Neoadjuvant chemotherapy is used as the primary treatment for locally advanced breast cancer to reduce tumour size and protect against metastatic spread. Monitoring the response to chemotherapy is achieved mainly by clinical assessment, which may prove unsatisfactory. This proof-of-principle study is to determine if changes of size and texture in the elastogram can track tumour response.A young woman with an invasive ductal carcinoma, visible on mammography and ultrasound (US), was treated with seven cycles of chemotherapy. Diagnostic mammography, MRI and core biopsy studies were performed. B-mode US and elastography datasets were collected in two planes prior to each cycle using a coarse calcification within the tumour as a constant reference point. Seven sequential strain images, each 3 weeks apart, were collected in total. The ratio of tissue strain within and outside the mass was calculated as an indicator of tumour response. The findings were compared with the post-chemotherapy MRI and mastectomy histology.The tumour showed almost complete histological response to chemotherapy. Sequential elastography studies demonstrated significant ratio changes in stiffness in tumour and peritumoral areas.Preliminary results suggest the changes in tumour and peritumour stiffness caused by chemotherapy are detectable by ultrasound elastography. Further investigation is required to evaluate the potential of elastography as a monitoring tool for chemotherapy treatment.


A second grant given by the National Cancer Institute was incorrectly omitted from the Funding statement. The number of the second grant is: 1RO1 CA109478.


After varicella, VZV becomes latent in ganglionic neurons along the entire neuraxis. During latency, virus DNA replication stops, transcription is restricted, and no progeny virions are produced, indicating a unique virus-cell (neuron) relationship. VZV reactivation produces zoster (shingles), often complicated by serious neurological and ocular disorders. The molecular trigger(s) for reactivation, and thus the identity of a potential target to prevent it, remains unknown due to an incomplete understanding of the VZV-neuron interaction. While no in vitro system has yet recapitulated the findings in latently infected ganglia, recent studies show that VZV infection of human neurons in SCID mice and of human stem cells, including induced human pluripotent stem cells and normal human neural progenitor tissue-like assemblies, can be established in the absence of a cytopathic effect. Usefulness of these systems in discovering the mechanisms underlying reactivation awaits analyses of VZV-infected, highly pure (>90%), terminally differentiated human neurons capable of prolonged survival in vitro.Varicella zoster virus (VZV) is a highly neurotropic, exclusively human herpesvirus. Primary infection causes varicella (chickenpox), wherein VZV replicates in multiple organs, particularly the skin. Widespread infection  VZV has been used to infect: (1) cultures of human ganglia, (2) human neural cells maintained as xenografts in mice, and, most recently, (3) embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The ultimate development of a model in which VZV becomes latent in neurons, characterized by the absence of a virus-induced cytopathic effect (CPE) and limited VZV transcription will provide a system to study molecules that induce reactivation. Identification of such molecules promises to identify therapeutic targets to prevent virus reactivation, a cause of serious, sometimes fatal neurologic disease, especially in the elderly/immunocompromised populations. This review presents an overview of VZV configuration and viral gene expression in latently infected human ganglia, followed by a comparison of various model systems designed to produce an equivalent virus-host relationship in neurons in vitro. Primary infection with varicella zoster virus (VZV), a highly neurotropic, exclusively human herpesvirus, causes varicella (chickenpox), during which time VZV replicates in multiple organs, particularly the skin . After vin vivo is confirmed by the ability of VZV to kill tissue culture cells in vitro derived from any organ. After varicella, VZV becomes latent in ganglionic neurons. Unlike primary measles virus infection of humans, varicella infection does not appear to involve the brain and spinal cord, a notion supported by the absence of simian varicella virus (SVV) in the CNS of non-human primates [in situ hybridization studies have identified multiple VZV transcripts in latently infected human ganglia. State-of-the-art multiplex PCR technology, capable of detecting all 68 annotated VZV gene transcripts, revealed transcription of at least 12 VZV genes during latency [During varicella, productive VZV infection occurs not only in the skin, but also in visceral organs. Widespread infection primates . During primates , with gaprimates ,10,11. A latency , of whic latency . Importa latency . Promote latency . Immunoh latency , results latency , thus rein vitro studies of the VZV-neuron relationship used VZV infection of explanted human brain and ganglia cells. Because most cells in these cultures were not neurons, VZV infection was productive. Interestingly, large intracytoplasmic vacuoles were seen in VZV-infected brain and ganglionic cultures, but not in control VZV-infected fibroblasts [The first roblasts . Human froblasts . Once agroblasts . Furtherroblasts . A recenroblasts  showed tHuman neural stem cells isolated from fetal brain remain viable and are capable of differentiation after transplantation into brains of non-obese, diabetic, severe combined immunodeficient (SCID) mice . At 4–6 Neuroblastoma cell lines are derived from human sympathetic nervous system tumors, which often originate in nerve tissues in the abdomen, chest and neck. Differentiation of the neuroblastoma cell line SH-SY5Y into neurons that contain at least one neurite-like projection when examined by phase-contrast microscopy results in cultures that are ~70% neuronal; most neuronal cells with projections stain positive for the neuronal marker synaptophysin . Infectiin vitro-fertilized human embryos at the blastocyst stage (first week of development) and can be maintained undifferentiated for at least 8 months (32 passages) in culture [Human embryonic stem cells (hESCs) are derived from the inner cell mass of  culture . hESCs c culture . Interes culture . HoweverhESC-derived neurons can be infected transaxonally, as evidenced by infection of neurons maintained in microfluidic chambers. These chambers contain two culture wells joined by 8- to 12-μm channels. Neurons are seeded into one well of the chamber, and medium with excess nerve growth factor (NGF) is placed into the second chamber, which stimulates axonal growth through the channels towards the chamber with the greater NGF concentration ,29. AddiHuman neuronal stem cells (hNSC) derived from fetal brain at 9 weeks’ gestation have also been used to study the VZV-neuronal relationship. Infection of hNSCs that were ~90% neuronal  with cell-free VZV (Zostavax vaccine) at a low MOI (~0.0025) revealed no CPE, and viral DNA, transcripts and protein (gE) were found 2 weeks later . Transfein vitro and yields cells that are ~80% βIII-tubulin-positive, a minority of which are also positive for the Brn3a and peripherin markers of sensory neurons [Induced pluripotent stem cells (iPSCs) are fibroblasts that are de-differentiated to mimic embryonic stem cells and then induced to differentiate into neuronal cells. This process requires at least 2 months  neurons . After iiPSCs are also available commercially. iCell neurons  are >95% βIII-tubulin-positive. Two weeks after infection with cell-free VZV (Zostavax vaccine), neuronal cultures appear viable, do not exhibit a CPE  or releaUltrastructural analysis of infected iPSCs  shows nuQuantitative RT-PCR and confocal microscopy show that gC transcripts and protein are greatly diminished in neurons compared to levels in productively infected fibroblasts , suggestHuman neuronal progenitor cells can be maintained for at least 6 months on inert microspheres kept in suspension within a NASA-designed rotary vessel. Similar to human trigeminal ganglia removed at autopsy , these cUnlike adherent neuron cultures, NHNP-TLAs survive for at least 180 days in culture. Infection with cell-free VZV results in replication of virus DNA and transcription of viral genes for ~18 days, after which a plateau is reached. Importantly, the infected cultures survive for 3 months. Despite their sporadic release of low amounts of infectious virus, NHNP-TLAs provide an opportunity to study the relationship of VZV with human neurons long-term.in vitro system has recapitulated findings in latently infected ganglia, recent studies indicate that VZV infection of neurons in vitro can be established in the absence of a cytopathic effect (in vitro. While in vitro latency has not been established, non-productive models of VZV infection in human neurons will be useful in elucidating the molecular events leading to virus reactivation in vivo.While no c effect , thus ho


We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML)  Varicella-zoster virus (VZV), the cause of varicella and zoster, is a human herpesvirus that replicates in the host cell nucleus where viral genomes are packaged into virion nucleocapsids. We have recently identified antiviral PML (promyelocytic leukemia) nuclear cages that sequester VZV nucleocapsids and inhibit formation of infectious particles. Here we developed a novel three-dimensional (3D) imaging and reconstruction strategy, termed Serial Section Array-Scanning Electron Microscopy (SSA-SEM) that together with electron tomography made it possible to derive 3D reconstructions of complete herpesvirus infected host cell nuclei and of PML cages with ultrastructural precision for the first time. We determined the 3D distribution of several thousand nucleocapsids within reconstructed volumes of single host cell nuclei and in PML cages as well as their sequestration efficiency and sequestration capacity: more than 98% of nucleocapsids were entrapped within PML cages and individual PML cages could sequester nearly 3,000 nucleocapsids which were cross-linked by an irregular electron-dense meshwork within the PML cages. This 3D analysis provides a proof of concept for using SSA-SEM to investigate virion assembly at the whole cell level and further elucidates our observation that PML cages are antiviral nuclear domains which block VZV nucleocapsid egress from the infected cell nucleus. During VZV infection, genome copies are synthesized in nuclear replication compartments and genomic DNA is packaged into icosahedral nucleocapsids formed by ORF40, the major capsid protein, and smaller capsid surface proteins, such as ORF23 protein. After assembly, nucleocapsids egress across the nuclear membrane for secondary envelopment in the cytoplasm and are then released as enveloped infectious virus particles PML protein has many different isoforms and is a major organizing component of these nuclear domains, which vary in shape, size, and molecular composition. PML isoforms share a conserved N-terminus, which is involved in PML oligomerization and contains a characteristic RBCC/TRIM motif. Different PML isoforms have unique C-terminal domains, which may be important in isoform-dependent functions in vivo by a mechanism involving the interaction of SUMO-interacting domains (SIM) of the VZV immediate early protein ORF61 with sumoylated PML protein in vivo in human skin xenografts and depletion of PML protein enhances VZV replication in cell culture, indicating a role for PML in the host cell defense PML-NBs have been implicated in controlling the replication of several alphaherpesviruses in vivo and in cultured cells in vitroIn the case of VZV, we found that if PML nuclear bodies are not dissociated effectively, these structures function to sequester VZV nucleocapsids in differentiated human cells within DRG and skin xenografts Quantitative immuno-electron microscopy analysis of ultrathin sections indicated that the majority (about 95%) of VZV nucleocapsids were found in PML cages, suggesting a surprisingly high efficiency of PML mediated capsid sequestration The goal of this study was to develop an EM imaging method with a high enough resolution to precisely identify, locate and count VZV nucleocapsids and at the same time, allow the efficient 3D reconstruction of large volumes of host cell nuclei, including complete PML cages. Here we describe a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM). Using this method together with electron tomography, we were able to create 3D reconstructions of complete nuclei of herpesvirus-infected cells and of PML cages with sequestered VZV nucleocapsids. Determining the shape and the volume of host cell nuclei and PML cages together with the precise 3D localization of several thousand VZV nucleocapsids enabled us for the first time to quantitatively estimate the sequestration capacity and efficiency of individual PML nuclear cages. The application of this strategy to resolve questions about PML-NB entrapment of VZV nucleocapsids is a proof of concept for its use to address other questions in virology and cell biology.As we have shown previously VZV-infected cell nuclei with diameters of about 5–10 µm and PML cages of 0.5-5 µm diameter We developed SSA-SEM as a method that provided both a high enough resolution to identify and precisely locate virion capsids and at the same time allowed the efficient 3D reconstruction of large volumes of host cell nuclei and complete PML cages . SSA-SEM3. The 3D reconstruction revealed an irregular shape of the nucleus characterized by several indentations and deep invaginations  mature and 316 (74.4%) immature nucleocapsids were identified and 102 (7.6%) mature and 1,238 (92.4%) immature capsids were identified in the nuclear volume in 3)  . Similar3) were not aggregated together with the other nucleocapsids (yellow spheres)  . 3,056 nPML cage . Of notePML cage .3 and contained four distinct PML cages (3 (3 (N = 4)  through a nucleus . Both thML cages  of very cages  of this compartment by SSA-SEM; however the 3D distribution of PML protein within PML cages was not revealed using this approach. Therefore we used a serial section immunoTEM (ss-immunoTEM) approach to investigate quantitatively and in three dimensions how PML protein is distributed within the shell and in the core of the PML cages, where the nucleocapsids are entrapped. Seven consecutive sections (100 nm) through HPF/FS-treated and LRwhite embedded cells that contained PML cages with entrapped VZV capsids, were labeled with a PML specific antibody and Protein A conjugated to 15 nm gold particles, and then imaged by TEM . The resThe observation by ss-immunoTEM that PML protein was present in the shell and in the center of PML cages, where it was found directly associated with many VZV capsids, suggested that PML protein is not only a structural component of the electron dense shell of PML cages, but may also be involved in the immobilization or cross-linking of sequestered VZV nucleocapsids. To address this hypothesis, we investigated the ultrastructure of PML cages and of sequestered nucleocapsids by electron tomography, which provided a higher resolution than SSA-SEM, albeit at the cost of allowing analysis of only a much smaller (thinner) sample volume. The samples for tomography consisted of HPF/FS-treated and epoxy resin-embedded VZV infected melanoma cells that expressed PML IV together with endogenous PML In this work, we developed Serial Section Array-Scanning Electron Microscopy (SSA-SEM), a novel three-dimensional (3D) imaging and reconstruction strategy, and applied the technique to the analysis of VZV-infected cells. Using SSA-SEM and EM tomography, we have reconstructed the nuclei of host cells infected with this representative herpesvirus and, for the first time, revealed the numbers and precise location of thousands of VZV nucleocapsids, visualized the 3D shape and ultrastructure of nuclear PML cages that entrap nucleocapsids, and provided quantitative estimates of the volume, sequestration efficiency and sequestration capacity of these PML cages. The large volume reconstruction of nuclei in VZV-infected cells also provided basic information on how VZV infection affects the shape of the host cell nuclei and how subnuclear domains like electron dense heterochromatin or PML cages and nucleocapsids are spatially related. Of interest, our 3D analysis revealed that PML cages with entrapped capsids were consistently located at the periphery of the nucleus and associated with domains of electron dense heterochromatin, suggesting that the formation of PML cages and VZV capsid sequestration are initiated adjacent to these domains.Our experimental challenge, which has many similarities to obstacles encountered in addressing other virology and cell biology questions, consisted in how to combine an efficient approach for the large volume 3D reconstruction of infected cell nuclei and complete PML cages with the high ultrastructural resolution necessary to localize VZV nucleocapsids and differentiate mature from immature capsids. Infected cell nuclei have diameters of about 5–10 µm and PML cages are about 0.5–5 µm et al. used SBF-SEM to locate structures reminiscent of actin filaments and nucleocapsids in cells infected with HSV-1 and PRV, which have caspids that are similar to VZV capsids in size and shape et al. used FIB-SEM to locate human immunodeficiency virus (HIV) particles in surface-connected tubular conduits in HIV-infected macrophages SSA-SEM combines a sample preparation strategy  similar to the method used in immunofluorescence (IF) array tomography with imaging and detection principles (high resolution SEM with back scattered electron detection) that have been used in serial block face (SBF)-SEM or focus ion beam (FIB)-SEM Our 3D reconstructions confirmed that most PML nuclear bodies in VZV infected cells expressing endogenous PML are disassembled efficiently during the course of infection. This process involves the interaction of SUMO-interacting domains (SIM) of the VZV ORF61 protein with sumoylated PML in vivo, nucleocapsids become sequestered in PML cages. Systematic random sampling analysis of hundreds of ultrathin sections through different PML cages suggested that >95% of all types of VZV nucleocapsids  were efficiently sequestered in PML cages However, when PML disassembly is incomplete, as it is in VZV-infected cells in skin and neural cells in vitro. In contrast, in the human host, VZV must infect complex tissues and overcome the barriers of intrinsic and adaptive immunity, which is likely to depend on production of larger numbers of infectious virus particles. Therefore the PML-mediated nuclear sequestration of many VZV capsids observed in human skin or DRG may be expected to have a more substantial antiviral effect in vitro is not just a tissue culture phenomenon.Given the high sequestration capacity of PML cages, now established by single 3D nuclear analysis and by quantitative random sampling analysis of hundreds of ultrathin cross-sections, it is somewhat surprising that infectious VZV titers were reduced only by about 50% in cell lines expressing PML IV Using conventional EM tomography, we obtained the first insights about the 3D ultrastructure of PML cages, suggesting how VZV nucleocapsids may be kept entrapped in these nuclear domains. Tomographic 3D reconstructions revealed the presence of an electron dense meshwork surrounding sequestered nucleocapsids and fiber-like like structures, that often cross-linked adjacent nucleocapsids, suggesting that capsids were entrapped by restricting their mobility and ‘gluing’ them together. The 3D analysis of PML-labeled sections by serial section immunoTEM showed that PML protein was present both in the periphery of the cage (the ‘shell’) and associated with the capsids entrapped in the center of PML cages. PML protein which is the main structural component of PML nuclear bodies, forms homo-and heterooligomers Cryo-tomography is an alternative that would enable a 3D reconstruction of PML cages at even higher resolution and more native conditions  but this approach can be predicted to encounter major experimental challenges. Frozen-hydrated sections through cell nuclei will be required, the identification of PML cages will be very demanding as these structures are not abundant, and identification would need to occur at a very low electron dose that creates noisy imaging conditions. Molecular docking of known crystal structures to electron densities will also be difficult, because of the many other proteins present in PML nuclear domain and currently only the PML RING domain has been crystallized in vivo and represents a novel antiviral mechanism, distinct from the established role of PML in controlling several alphaherpesviruses shortly after virus entry by limiting early viral gene transcription. Visualization of the shape and measurements of the volumes of host cell nuclei and PML cages together with the 3D localization of VZV nucleocapsids with ultrastructural precision enabled us to determine the sequestration efficiency and capacity of PML nuclear cages. This work contributes not only to a more comprehensive understanding of the antiviral activity of PML cages against VZV, a pathogenic human herpesvirus, but also provides a novel method to undertake the 3D reconstruction and quantitative investigation of nuclear PML domains that have also been found to be associated with capsids of papillomaviruses and polyomaviruses In summary, we were able to create complete reconstructions of herpesvirus-infected cell nuclei and PML nuclear domains in three dimensions for the first time using 3D SSA-SEM and EM tomography. This study supports and extends our recent discovery and characterization of PML cages that efficiently sequester VZV nucleocapsids in cell culture and in differentiated human skin and neural cells infected The human melanoma cell line  was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, nonessential amino acids (100 µM) and antibiotics (penicillin at 100 U/ml and streptomycin at 100 µg/ml). Melanoma cells were passaged fewer than 25 times. Melanoma cells expressing doxycyline-inducible PML IV were constructed using the pRetro-X-Tet-On-Advanced vector system and pRetro-X-Tight-Pur plasmid (Clontech Laboratories) with the PML IV plasmid pcDNA3-PML IV as described recently SP2 confocal laser scanning microscope . Microscope objectives were 40×/1.0  or a 63×/1.4 (N.A.) Plan Apochromat objectives. Images were scanned at 1024×1024 pixels with at least four times frame averaging and the pinhole adjusted to one airy unit. Brightness and contrast were adjusted using Photoshop CS3 (Adobe) or iPhoto (Apple).Cultured cells on glass coverslips were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were blocked and immunostained as described previously For standard TEM, samples were fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (ph 7.2) and embedded in epoxy-resin. For immuno-TEM or EM tomography samples were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (ph 7.2) and then high-pressure frozen (HPF) in a Leica EM PACT2 and freeze substituted (FS) in either LR-white or Epoxy resin (Embed812), respectively. Frozen specimen carriers with cells were placed into frozen cryovials containing acetone with 0.1% glutaraldehyde and 0.1% uranyl acetate (for LR White embedding) or in acetone with 1% osmium tetroxide and 0.1% uranyl acetate (for Epon embedding). The frozen vials were then placed into a Leica AFS for the freeze-substitution procedure and then embedded in either or LR-White resin for immuno-TEM or epoxy resin Embed 812 for EM tomography. Sections (80–300 nm) were prepared with a diamond knife (Diatome) using an ultramicrotome . For immunogold-labeling LRwhite sections were pre-blocked in DIG-blocking solution (Roche) for 30 min. Primary antibodies and Protein A-gold particles  were diluted in blocking solution and sections were incubated for 1 h or 30 min, respectively, at RT. Rabbit polyclonal anti-PML antibody (Santa Cruz Biotech) was used at 1∶10 dilution. Sections were stained with 3.5% aqueous uranyl acetate for 15 minutes and with 0.2% lead citrate for one minute and air-dried. Sections were analyzed using a JEOL 1230 transmission electron microscope (TEM) at 80 kV and digital photographs were captured with a GATAN Multiscan 701 digital camera.VZV-infected cells were fixed in 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 hr and cell pellets were stabilized by embedding in 10% gelatine. The samples were washed with ultrapure water and then postfixed with 2% osmium tetroxide reduced with 1.5% (w/v) potassium ferrocyanide for 2 hr at room temperature. The samples were then washed again, followed by one hour incubation with 1% (w/v) tannic acid, washing in ultrapure water and a final “en block” staining with 3.5% (w/v) uranyl acetate over night. The samples were then dehydrated in a series of ascending ethanol concentrations (30%–100%), treated with propylene oxide and finally embedded in epoxy resin Embed 812 . The procedure for the preparation of Serial Section Arrays (SSA) was similar to the method described for fluorescence array tomography The arrays were first pre-scanned with a Hitachi S-3400N VP-SEM to assess the quality of the ribbons of serial sections and to find regions of interest (ROIs). ROIs were mapped at a magnification from 100× (whole section image) to 10,000× (image of cell nucleus with resolved capsids) using an accelerating voltage of 10 kV, a working distance of 8.5 mm and the back-scattered electron (BSE) detector. For the final acquisition of high-resolution digital image stacks from serial sections, the arrays were transferred to a Zeiss Sigma FE-SEM that is equipped with a field emission gun (FE). The mapped ROIs were identified and then imaged using the BSE detector at magnifications from 5,000–40,000× with an accelerating voltage from 6 kV–10 kV and a working distance from 6–7 mm; images were scanned at 2048×1536 pixels, with at least 2 times line averaging.http://fiji.sc/wiki/index.php/Fiji). Aligned images were saved as image sequence files and then imported into the 3D reconstruction program ‘Reconstruct’(http://synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm) SSA-SEM image stacks were automatically aligned (registration) in rigid mode using the ‘StackReg’ plugin in the Fiji/ImageJ software package  using a tilt range of ±65° at 1.5° angular increments VZV infected cells were fixed and high pressure frozen (HPF), freeze substituted (FS) and embedded in epoxy resin (Embed812) as described above (TEM sample preparation). 80 nm or 300 nm sections were cut with a diamond knife (Diatome) using an ultramicrotome  and placed on Formvar and carbon coated 75mesh TEM copper grids (TedPella). Sections were stained with 3.5% aqueous uranyl acetate for 15 minutes and with 0.2% lead citrate for one minute and air-dried. Finally, the sections were coated on both sides of the grid with 15 nm colloidal gold particles (Ted Pella) as fiducial markers by repeated dipping of the grids in the colloidal gold solution followed by air drying. The 80 nm thick sections were imaged on a JEOL 1400 TEM  at 120 kV equipped with a dual-axis tomography holder. The double-tilt series were recorded with the ‘SerialEM’ software package (http://bio3d.colorado.edu/imod/) The series of dual-axis tilt images were aligned, reconstructed by weighted back-projection and then merged into dual-axis tomograms using the software package IMOD 4.1 (Graph Pad Prism (version 5.0) statistical software was used for quantification and statistical analysis.Video S1Corresponds to (AVI)Click here for additional data file.Video S2Corresponds to (AVI)Click here for additional data file.Video S3Corresponds to (AVI)Click here for additional data file.Video S4Corresponds to (AVI)Click here for additional data file.Video S5Corresponds to (AVI)Click here for additional data file.Video S6Corresponds to (AVI)Click here for additional data file.Video S7Corresponds to (AVI)Click here for additional data file.Video S8Corresponds to (AVI)Click here for additional data file.Video S9Corresponds to (AVI)Click here for additional data file.Video S10Corresponds to (AVI)Click here for additional data file.Video S11Corresponds to (AVI)Click here for additional data file.Video S12Corresponds to (AVI)Click here for additional data file.Video S13Corresponds to (AVI)Click here for additional data file.Video S14Corresponds to (AVI)Click here for additional data file.


An organization providing funding the grant to the last author (JC) were incorrectly omitted, and a funding organization for that grant was also incorrectly given in the Funding Statement. The first sentence of the Funding Statement should read: “This work was supported in part by the grant from the Foundation for Polish Science TEAM/2010-5/1, co-financed by the European Union within European Regional Development Fund and Polish National Science Center grant 2011/02/A/NZ5/00337 (to JC)."


Understanding the multiple biological functions played by human mesenchymal stem cells (hMSCs) as well as their development as therapeutics in regenerative medicine or in cancer treatment are major fields of research. Indeed, it has been established that hMSCs play a central role in the pathogenesis and progression of tumours, but their impact on tumour growth remains controversial.+) to obtain subcutaneous or lung tumours. When tumour presence was confirmed by non-invasive bioluminescence imaging, hMSCs were injected into the periphery of the SC tumours or delivered by systemic intravenous injection in mice bearing either SC tumours or lung metastasis.In this study, we investigated the influence of hMSCs on the growth of pre-established tumours. We engrafted nude mice with luciferase-positive mouse adenocarcinoma cells (TSA-LucRegardless of the tumour model and mode of hMSC injection, hMSC administration was always associated with decreased tumour growth due to an inhibition of tumour cell proliferation, likely resulting from deep modifications of the tumour angiogenesis. Indeed, we established that although hMSCs can induce the formation of new blood vessels in a non-tumoural cellulose sponge model in mice, they do not modify the overall amount of haemoglobin delivered into the SC tumours or lung metastasis. We observed that these tumour vessels were reduced in number but were longer.Our results suggest that hMSCs injection decreased solid tumour growth in mice and modified tumour vasculature, which confirms hMSCs could be interesting to use for the treatment of pre-established tumours. Under appropriate experimental conditions, MSCs can differentiate into a number of mesodermal cell lineages, including bone, cartilage, stroma, adipose tissue, connective tissue, muscle and tendon [Mesenchymal stem cells (MSCs), also referred to as stromal progenitor cells, reside in the adult bone marrow, are capable of self-renewal and can be quickly expanded d tendon -3. MSCs d tendon .Moreover, MSCs possess an innate tropism for sites of injury irrespective of tissue or organ type. MSCs have been shown to specifically home to tumour and metastasis sites in multiple types of cancers . The troin vitro[in vivo[However, the role of native MSCs inside the tumour microenvironment is unclear, and the relationship between MSCs and tumour cells is complicated; MSCs have been linked to contradictory effects on tumour growth -11. Thisin vitro,13 and io[in vivo-16. In ao[in vivo,18 and/oo[in vivo. In someo[in vivo and an io[in vivo. In conto[in vivo.in vivo, these cells do not alter the overall pathological angiogenesis of the tumours. However, administration of hMSCs induced significant remodelling of the tumour vasculature. These observations suggest that tumour vasculature normalisation is induced by the presence of hMSCs.To clarify this issue, we studied the impact of human mesenchymal stem cells (hMSC) injection on pre-established tumours. We demonstrated that hMSC administration decreases tumour growth when injected systemically or directly in contact with the tumours, suggesting a systemic effect. This effect is clearly correlated with an inhibition of tumour cell proliferation. Furthermore, we established that although hMSCs induced physiological angiogenesis 2 incubator in RPMI 1640 media supplemented with 1% glutamine, 10% foetal bovine serum, 50 units/ml penicillin, 50 μg/mL streptomycin, 25 μM β-mercaptoethanol and 700 μg/ml Geneticin™ .TSA-pGL3 is a cell line derived from the murine mammary adenocarcinoma TS/A-pc cell line comes from spontaneous mammary tumour cells  that has2 according to a previously described method [in vitro differentiation in adipocyte and osteoblast [hMSCs were isolated from bone marrow aspirates from three healthy donors, each of whom gave informed consent. All the isolation and culture procedures were conducted in the authorized cell therapy unit  at the Grenoble University Hospital. The cells were cultured at 37°C in a humidified atmosphere containing 5% COd method . Brieflyteoblast ) (data n6 TSA-pGL3 cells suspended in 200 μL of PBS into the right flank of mice resulted in the formation of 6 to 8 mm-diameter tumours after one week. Intravenous (IV) injection of 105 TSA-pGL3 cells suspended in 200 μL of PBS into the tail vein resulted in tumour lung nodule formation at day 4. At these times, animals were randomised into groups (n = 5 per group), and 5 × 105 hMSCs were injected subcutaneously at the tumour site or intravenously.All animal experiments were conducted in adherence to the Principles of Laboratory Animal Care  and approved by the regional ethics committee ). Female athymic NMRI nude mice, purchased from Janvier  at 6 to 8 weeks of age, were maintained under specific pathogen-free conditions. We choose to inject the TSA-pGL3 cell line (1) subcutaneously because it is an easy model to follow and visualize and (2) intravenously to mimic a more physiological lung metastasis model   was again injected into the sponges through the skin on day 2 and 3. At 7 days after implantation, the mice were anesthetised, and the sponges were rapidly excised and photographed. Each sponge was then homogenised in 1 mL RIPA lysis buffer with protease inhibitors, and the supernatants were subjected to haemoglobin quantification using Drabkin’s reagent , expressed as mg/ml.Cellspon cellulose sponges  were implanted under the skin of NMRI nude mice . Operatiet al. [All imaging was performed under inhalational anaesthesia (3% isoflurane) and administered to a free breathing mouse using a nose cone. For bioluminescence imaging, mice received an intraperitoneal injection of D-luciferin potassium salt dissolved in sterile phosphate-buffered serum (150 mg/kg) 5 min before imaging , as described previously by Jin et al. ,28. Semi4 , which targets the integrin αvβ3, was injected intravenously through the tail vein of each mouse. Three-dimensional fluorescence acquisition and quantification were performed 16 h post-injection for the tumour models and 3 h for the sponge model with the continuous-wave fluorescence-enhanced diffuse optical tomography system previously described by Koenig et al. [2 field of view) over the region of the mouse where the tumour is implanted. Two scans were successively performed for diffusion and fluorescence. The exposure time was automatically computed at each laser position to use the entire dynamic range of the camera. The two stacks of diffusion and fluorescence images were analysed by the reconstruction algorithm to generate a three-dimensional image [et al. [For three-dimensional fluorescence imaging, 200 μl Alexa700-RAFT-c(−RGDfK-)g et al. ,30. fDOTal image . The fDO [et al. .2O2 in methanol for 20 min. Sections were then sequentially incubated for 1 h with a rat monoclonal anti-CD31 antibody , rabbit anti-Ki67  or rabbit anti-alpha smooth actin antibody  and for 1 h with goat anti-rat  or goat anti-rabbit  secondary antibodies, as appropriate. Peroxidase activity was revealed using diaminobenzidine tetrachloride as a chromogen . Sections were counterstained with haematoxylin and mounted.Frozen sections (8 μm) from subcutaneous tumours were fixed in acetone for 10 min. Sections were then washed three times for 5 min each in Tris-buffered saline containing 0.1% Tween-20 and endogenous peroxidases were blocked with 0.1% HImmunohistochemical staining against the endothelial marker CD31 was followed by observation under a low magnification scope (100×) for five fields of view of each tumour (five tumours by condition). Then, vessel length and quantity were measured in each of these areas using ImageJ software . All couAfter immunohistochemical staining against Ki67 and αSMA, slides were observed under a high magnification microscope (200×). For Ki67, six to nine areas were photographed for each tumour. For αSMA, two to nine areas were photographed for each tumour. These photographs were analysed using the ImmunoRatio plug-in from ImageJ software . The Ki62, 200 μM dNTPs, 400 nM each primer, 0.5 U Taq polymerase . The PCR conditions were: step 1, 95°C for 30 sec, step 2, 62°C for 30 sec, step 3, 72°C for 30 sec. To ensure semi-quantitative results in the RT-PCR assays, the number of PCR cycles was selected to be in the linear range of amplification for each set of primers (n = 30 for GAPDH and n = 35 for hCD90). PCR products were separated and visualised after electrophoresis on 2% agarose gels containing 1 μg/ml ethidium bromide.Total RNA was extracted from tumours and organs with TRIzol™ . The total RNA was quantified using a Nanodrop ND-2000 instrument  and reverse transcription was performed on 1 μg total RNA with SuperScript III™ RNaseH reverse transcriptase  under the conditions recommended by the manufacturers. Before PCR, quantities of cDNA samples were adjusted to yield equal amplifications of the mRNA encoding the housekeeping gene GAPDH . PCR for GAPDH and human CD90  were performed in a final volume of 25 μl containing 1 × PCR buffer, 2 mM MgClt test. Statistical significance was assumed when P <0.05.Statistical analyses were performed using StatView™ . All results are expressed as the mean ± standard deviation. Comparisons between groups were performed using a two-tailed Student’s et al. [P = 0.03) or by the IV route . This also corresponded to a slight decrease in tumour volumes . In theP = 0.0264) smaller bioluminescence signal than the control mice . The anti-proliferation effect was not significantly different when the hMSCs were injected systematically or near the tumour site, suggesting that the inhibitory effect of hMSCs on tumour cell growth could be indirect. Indeed, one day after hMSCs IV injection, human CD90 mRNA expression was detected by RT-PCR in different tissues and organs, including subcutaneous tumours  and TSA-pGL3 + hMSCs tumours . Similar results were obtained when we quantified the Hb content in the sponges instead of using the RGD-based assay. Again, hMSC treatment was as pro-angiogenic as FGF-2 treatment (3.094 mg Hb/mL ± 0.816 for hMSCs and 2.939 mg Hb/mL ± 0.824 for FGF-2)  by three-dimensional Alexa700-RAFT-c(−RGDfK-)To determine whether the influence of hMSCs on tumour growth could be related to the pro-angiogenic properties of the cells, we evaluated the vascularisation of the tumours after hMSC inoculation.In vivo imaging of subcutaneous or lung tumours using Alexa700-RAFT-c(−RGDfK-)4 was performed  or IV (P = 0.0125)) compared with mice injected with tumour cells alone. We observed not only a decrease in the number of tumour vessels but also an increase in vessel length. Indeed, the median vessel length was 85 μm for tumour cells alone and 323 μm (P <0.0001) and 290 μm (P = 0.0016) when hMSCs were injected SC or IV. In addition, analyses of vessel maturation were performed by αSMA labelling (pericyte staining) in sections from subcutaneous TSA-pGL3 (control) and TSA-pGL3 + hMSCs tumours  and 7.55 ± 4.51% (P <0.0001), when hMSCs were injected at the tumour periphery or IV, respectively.We then determined whether the vessel structure was different in the presence of hMSCs. Sections of subcutaneous tumours were stained with an antibody directed against CD31 to visualise the blood vessels .The effect of inhibition on tumour growth is clearly correlated with a decrease in the Ki67 labelling index. Our hypothesis is that this effect could be indirect because we observed the same result when hMSCs are injected by means of the IV route or directly beside the tumour site. Moreover, these results are obtained even when hMSCs are not or only poorly and transiently detected at the tumour site. In our model using nude mice, hMSCs were detected by RT-PCR one day after IV injection in the tumour as well as in the lung, liver and spleen, as previously described -44, but in vivo physiological model of angiogenesis based on the introduction of a cellulose sponge template under the skin of the mouse and demonstrated the pro-angiogenic activities of hMSCs. Indeed, the in vivo angiogenic effect of hMSCs is as strong as the effect of basic FGF-2. This result was consistent with published data from another angiogenic, but less physiological, model of in vivo Matrigel implantation [et al. [in vitro and a diminution of B16 tumour growth but did not analyse the structure of the tumour blood vessels.Because different studies have demonstrated that MSCs play a role in angiogenesis, we decided to evaluate the influence of hMSCs on tumour angiogenesis. In a first assay, we used an antation -16. Howeantation ,52. It iantation . We alsoantation . In paraantation  and thatantation ,57. It w [et al.  using MSin vivo, these cells do not alter the overall tumour angiogenesis. However, administration of hMSCs induces significant remodelling in tumour vasculature.In this study, we demonstrated that hMSCs administration decrease pre-established tumour growth, when hMSCs were injected systemically or directly into contact with the tumour. This effect is clearly correlated with an inhibition of tumour cell proliferation. More, whereas we have shown that the hMSCs induced angiogenesis In summary, our results confirm that MSCs could be interesting to use for the treatment of pre-established tumours, particularly in combination with other therapies. However, the complex mechanisms of hMSC action remain to be characterised further.FCS: Foetal calf serum; Hb: Haemoglobin; hMSC: Human mesenchymal stem cell; IV: Intravenous; PBS: Phosphate-buffered saline; RT-PCR: Reverse-transcriptase polymerase chain reaction; SC: Subcutaneous; SD: Standard deviation; TSA-pGL3: TS/A-pc mouse adenocarcinoma cell line stably transfected with the luciferase reporter gene.The authors declare that they have no competing interests.MK, FDF, AK and CR were involved in the practical achievement of these experiments. Human stem cells for clinical use were obtained and characterized by AM, VP, FDF and MJR. Collection, analysis and interpretation of the data as well as the redaction were performed by CR, MK, FDF and JLC. Financial and administrative support was carried out by JLC and CR. All authors have read and approved the manuscript for publication.


The Stokes flow induced by the motion of an elastic massless filament immersed in a two-dimensional fluid is studied. Initially, the filament is deviated from its equilibrium state and the fluid is at rest. The filament will induce fluid motion while returning to its equilibrium state. Two different test cases are examined. In both cases, the motion of a fixed-end massless filament induces the fluid motion inside a square domain. However, in the second test case, a deformable circular string is placed in the square domain and its interaction with the Stokes flow induced by the filament motion is studied. The interaction between the fluid and deformable body/bodies can become very complicated from the computational point of view. An immersed boundary method is used in the present study. In order to substantiate the accuracy of the numerical method employed, the simulated results associated with the Stokes flow induced by the motion of an extending star string are compared well with those obtained by the immersed interface method. The results show the ability and accuracy of the IBM method in solving the complicated fluid-structure and fluid-mediated structure-structure interaction problems happening in a wide variety of engineering and biological systems. There are abundant scientific and engineering applications where a flexible structure is immersed in a viscous incompressible fluid. The motion of the structure can induce the fluid flow motion and vice versa. The combination of the fluid and structure motions constitutes the so-called fluid-structure interaction (FSI) problem  Stokes flow induced by motion of an extending star string (FSI); (ii) Stokes flow induced by motion of a filament fixed at one end (FSI); (iii) Stokes flow induced by motion of the same filament considered in the second test case, but now deforming a secondary elastic immersed boundary placed in the flow domain (fluid-mediated structure-structure interaction). In order to benchmark the numerical simulation carried out in the present work, the results of the first test case are compared with those obtained by the immersed interface method . The las2 denotes the Laplacian, p  is the pressure, and μ is the viscosity. It may be noted that The flow induced by the elastic boundary motion is assumed to be in the Stokes regime and hence it is governed by the Stokes equations. The Stokes flow refers to the highly viscous fluid flow, in the limit where the Reynolds number tends to zero and both the inertial and convection terms are omitted from the Navier-Stokes equations –22. In t∇·V→=0,(∇2u)+fx,0=−∂p∂y+μx and y and be added together to give the following relation:p is known,  can  can 3) celation:∇2p=∇→·f→s   indicates the Lagrangian coordinate along the boundary and t denotes time. According to the principle of virtual work, the Lagrangian force cs and cb are constants denoting the stiffness and bending coefficients, respectively. After substitution of E[X→E[X→f→ isthat is,F→=−∂E are  are 8) aδ denotes the Dirac delta function. In the present work, we use the delta function proposed in [Ω denotes the fluid domain to be influenced by the motion of the immersed boundary. The immersed boundary position is then updated by using the following equation:The transformation between the Eulerian and Lagrangian variables can be realized by the Dirac delta function . Spreadiposed in :(10)δh(k = 0) and ends at its fixed end (k = N).A staggered grid  is used in the Lagrangian coordinate system see , where tDs, Dss, and Dsss denote, respectively, the finite difference (FD) approximation to the first, second, and third order derivatives with respect to the arc-length s. For an arbitrary variable ψ, for instance, the following notations:T  and the tangent vector k = 0), the bending force term is discretized asSuppose that  τ→ (see ) can be filament :(18)={14πmations: δ(x)={14πh is the Eulerian grid spacing. Similarly, the discretized form of the force density is given byThe discretized form of the local fluid velocity at the structure position ondition , which iFn at Lagrangian points Xn using (Compute Lagrangian forces n using .Fn to Eulerian grid points using (Communicate Lagrangian forces ts using .u.Solve fluid equations from  to 3) t t3) to gU using  but could increase the time step at later times. Starting the work we compute velocity, pressure, Lagrangian, and Eulerian forces at the first time step. A grid size of 64 × 64 in a square domain with the dimensions of × is considered. The results have been shown in Figures In this test case, we study the motion of a nonequilibrium star string immersed in an incompressible fluid while relaxing to its circular equilibrium shape. The initial velocity and pressure of the fluid are set to zero, and the only driving force is the string tension. The fluid flow and the string motion are fully coupled. At equilibrium, the velocity is zero and the pressure is piecewise constant inside and outside the balloon. The initial shape of the distorted string is expressed in the cylindrical coordinates as8ρ=0.6+0.It can be seen from rminn and rmaxn behave computationally over a short-time scale.According to The instantaneous maximum and minimum radii of the boundary are obtained from the following relations:t = 0 are depicted in In this section, the Stokes flow induced by the motion of a bending filament is examined. An initial bending force is applied to the filament. Initially the fluid is at rest. After the first time step, the Lagrangian points representing the filament boundary move from their initial positions in the flow direction. This leads the points to become too close to (or too far away from) each other. To keep the inextensibility of the filament, a (relatively) large stiffness coefficient is applied. Under such conditions, only the angle between the nodes varies, but the distance between them remains unchanged. In addition, to circumvent the instability arising when proper tension and bending forces are not applied, very small time steps are considered. As shown in By inserting the Eulerian forces to the flow field equations and solving them, the velocity and pressure fields can be obtained at different time steps. The values of various constants considered for the case of the bending filament are given in x-component velocity along with the streamlines at different time steps. It can be seen that the motion of the filament from its initial nonequilibrium state (Frame (a) in The pressure contours associated with the different times shown in In this last test case, the motion of an initially bent filament in a square cavity causes the fluid motion, which in turn leads to the motion and deformation of an initially circular elastic boundary placed somewhere in the cavity. It may be noted that, for an immersed boundary to move in a fluid, either the fluid should have enough velocity to move the boundary or the initial deformation of the boundary itself would cause the fluid motion. In the present example, both of these mechanisms are present simultaneously.The secondary immersed boundary considered here is an elastic circular string, which is initially in its equilibrium state without any deformation or internal forces. For this boundary, the tension forces are applied to avoid the Lagrangian points representing the boundary to move too far from (or too close to) each other. In addition, no bending force has been applied to this boundary in order to allow the angle between the points to change freely. As shown in t = 0.001 have been depicted in For the circular-immersed boundary, the stiffness coefficient is set to be relatively small to allow the points to move easily far away from each other. At the outset of the calculations, this boundary exerts no force on the initially quiescent fluid domain. In fact, the fluid motion is induced by the motion of the filament, which is initially bent. The moving fluid can now exert forces on the circular boundary and move and deform it. The Lagrangian forces associated with the immersed boundaries and the consequent Eulerian forces imposed on the fluid domain by theses boundaries at time x-component velocity along with the streamlines on the one hand and the contours of pressure on the other hand at times t = 0.001, 0.0075, and 0.0165. The sequential deformations of the immersed boundaries are also included in this figure. The circular boundary is found to experience significant deformation as it moves upward . A single eddy forms inside the circle at time 0.0165. As expected, one can see from this figure that the pressure is discontinuous across both of the immersed boundaries.In the present work, the Stokes flow induced by the motion of an elastic massless body immersed in a two-dimensional fluid is studied using immersed boundary method. Initially, the immersed body is unstable and the fluid is at rest. The body can induce fluid motion while returning to its equilibrium (stable) state leading to a fluid-structure interaction problem. In the current study, two different test cases are examined. In both of the cases, the motion of a massless filament fixed at one end induces the fluid motion inside a square domain. However, in the second test case, a deformable circular string is placed in the square domain and its interaction with the Stokes flow induced by the filament motion is studied. In order to verify the accuracy of the numerical method employed, the simulated results associated with the Stokes flow induced by the motion of an extending star string are compared well with those obtained by the immersed interface method. The results show the ability and accuracy of the IBM method in solving the complicated FSI problems happening in a wide variety of engineering and biological systems.


Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells. Varicella-zoster virus (VZV), the alphaherpesvirus that typically causes childhood chickenpox and shingles in adults, becomes latent in neurons, thus remaining in the body for a lifetime. Unfortunately, few models are available to study the establishment of VZV latency since the virus infects only humans and establishes persistent infections and latency only in neurons, a slowly proliferating, short-lived cell in culture. We have successfully maintained normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs) in 3-dimensional (3D) cultures for up to 6 months. The 3D NHNP TLAs show some characteristics as those found in the human trigeminal ganglia, the site of VZV latency. NHNP TLAs infected with VZV remain viable for 3 months during which time VZV DNA replicates and remains genetically stable, virus genes are transcribed, and infectious VZV is sporadically released. The ability to maintain VZV infected NHNP cells in culture for extended times provides the unique opportunity to study the molecular interactions between this important human pathogen and neuronal tissue to an extent previously unattainable. L) and unique short (US), each flanked by inverted repeat regions  Varicella-zoster virus (VZV) is a ubiquitous human herpesvirus, as evidenced by a seroprevalence of more than 95% worldwide in vitro, (ii) the availability of human ganglia is limited, (iii) the time between death and tissue collection cannot be predetermined, (iv) there are considerable human to human variations including preexisting virus burden and time since primary VZV infection, and (v) since most ganglia explants contain latent VZV, it is difficult to perform prospective studies using VZV with defined genetic mutations. Therefore, our understanding of VZV latency largely represents a snapshot of human ganglia that show a high degree of variability VZV pathogenesis, latency, and reactivation are difficult to study, as the virus exclusively infects humans and no animal model is currently available to investigate VZV latency. As a result, our knowledge of VZV latency is based on the analysis of human ganglia removed at autopsy. However, primary human ganglia have several disadvantages: (i) they can only be cultured for a few days Human fetal dorsal root ganglia (DRG) are typically free of latent VZV. Enzymatically dissociated human DRG show neurite outgrowth when maintained in culture and have facilitated the analysis of apoptosis inhibition mediated by VZV in neurons Human stem cells provide an alternative source of neurons. Stem cell-derived neurospheres can be maintained in tissue culture and in lateral ventricles of neonatal non-obese diabetic SCID mice. While human neurospheres maintained in mice can be infected with VZV and show no obvious virus-induced cytopathic effect, time course experiments using this system are technically challenging and costly In order to overcome these problems, we developed a novel three-dimensional (3D) culture system to maintain partially differentiated normal human neural progenitor (NHNP) cells as tissue-like assemblies (TLAs) that share some features with neurons found in human trigeminal ganglia. The 3D NHNP TLAs can be maintained for at least three months following VZV infection. During this time, VZV DNA was readily detected, VZV immediate-early, early and late genes were transcribed, no VZV-induced CPE was observed, but low amounts of infectious virus were released into the culture medium. Importantly, the VZV genome was stable throughout the three month infection period, suggesting that the 3D NHNP TLA culture system may be useful to investigate the long-term interplay between VZV and neural tissue.in situ hybridization (mFISH), flow cytometry, confocal microscopy and RT-PCR to assess their genetic stability as compared with 2D NHNP cell cultures. Comparison of 2D and 3D cultured NHNP cells by mFISH respectively showed no chromosomal rearrangements or breaks, indicating genetic stability of NHNP TLAs for at least a six month period in culture (+ (+ (data not shown) were not detected in either culture. An example of the tissue-like complexity of the NHNP TLA constructs is shown by environmental scanning electron micrographs (ESEM) in NHNP cells were cultured in 3D on a scaffold of Cultispher beads in a rotating wall vessel (RWV) bioreactor and analyzed by multicolor fluorescence  culture . Flow cy culture . Markerslture , and confirming the suspected recombination. Growth kinetics showed no significant effect on virus replication due to insertion of the fluorescent proteins into ORF63/70  were mutated to identify expression originating from each gene. The fluorescent proteins eGFP and mRFP were inserted at the C-terminus of ORF63 and 70, respectively, in the parental VZV Oka strain. For genetic manipulations, an infectious BAC clone (pP-Oka) was used and the insertion of the marker genes resulted in recombinant BAC p63G/70R . CorrectORF63/70 . Taken tCell-free v63G/70R efficiently infected NHNP TLAs, as evidenced by an approximate 50-fold increase in VZV genome copies from 0 to 18 days post-infection (dpi). After 18 dpi, VZV genome copy numbers remained constant . To compde novo production and release of infectious virus  into a 55 ml rotating wall vessel bioreactor  and grown at 37°C under a 5% CO2 atmosphere. Cells were allowed to attach to the beads for 48 h in the bioreactor before re-feeding with GTSF-2 containing 10% FBS. To maintain the TLA cultures within normal human physiological blood chemistry parameters (pH 7.2 and a glucose concentration of 80–120 mg/dL), 20–90% of the media was replaced as required with fresh GTSF-2 media every 48 h, facilitating efficient TLA tissue growth and maturation prior to VZV infection. All metabolic determinations were made using an iStat hand held blood gas analyzer .NHNP cells were obtained from Lonza  and propagated in GTSF-2, a unique media containing glucose, galactose and fructose supplemented with 10% fetal bovine serum (FBS), at 37°C under a 5% CO2Human melanoma cells  were propagated in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin  at 37°C under 5% CO2, 10 mM Tris-HCl, pH 7.4). Cells were disrupted by Dounce (type A) homogenization , debris removed by centrifugation at 900×g for 15 min and virus containing supernatant filtered using a 1.0 µm Millex filter unit .Cell-free VZV 2 atmosphere at 37°C. Every 24 h.p.i., ∼80% of the culture media was replaced with fresh GTSF-2 containing 10% FBS. Samples were collected approximately every other day for ∼70 days to determine viral genome copies as described below. Supernatant of infected NHNP TLAs was removed, centrifuged to remove cells and debris and titrated in fresh MeWo cells to determine the amount of infectious VZV virions in the NHNP culture media.NHNP TLAs were infected in the RWV with cell-free VZV at a multiplicity of infection (MOI) of 0.1 by absorption at room temperature for 30 min in 20 ml GTSF-2. Then, the RWVs were filled with fresh GTSF-2/10% FBS and transferred to a humidified incubator with a 5% COMulticolor mFISH for the analysis of chromosome integrity was conducted as described Sample preparation, scanning and analysis of the TLAs was performed as described previously a6) and β-Tubulin-III at the following dilutions: CXCR4 50 µg/ml, CD133 25 µg/ml, Nestin 50 µg/ml, CD105 Endoglin 50 µg/ml, CD90 Thy-1 50 µg/ml, CD49f (ITGa6) 25 µg/ml, β-Tubulin-III 25 µg/ml, CD34+ 25 µg/ml, and CD38 25 µg/ml. All reagents were obtained from R&D Systems  with the exception of CXCR4 obtained through ABCAM . Confocal and flow cytometry used the same reagents and confocal analyses were performed as described previously g. The soft cell pellet was resuspended in GTSF-2 and permitted to recover for 1 h at 37°C. After recovery, viable cells (trypan blue exclusion) were counted and aliquots (∼1×105) were washed 2× in PBS and used for flow cytometry. Fluorochromes were added and incubated 45 min at 4°C in PBS, washed 2× in PBS at 4°C and brought to 400 µL with PBS. All analysis was done on freshly conjugated cells. Samples were either analyzed by confocal microscopy on a Leica TCS/SP2 3-laser confocal microscope or a Beckman Coulter XL2 Flow Cytometer , CD90 (Thy-1) CD49f . Harvest of cadaver tissues is not considered human research as determined by the IRB. Trigeminal ganglia were fixed in 4% paraformaldehyde overnight, dehydrated through graded ethanol, and embedded in paraffin. Five micrometer sections were collected on Superfrost  slides and fixed at 72°C for 30 min. Samples were deparaffinized in xylene and rehydrated in graded ethanol. Antigen was retrieved by soaking the sections in 10 mM sodium citrate (pH 6.0) in a steamer for 25 min. Slides were blocked in 5% normal goat serum for 1 h at room temperature and then incubated with mouse anti-Nestin monoclonal  or mouse anti-β-Tubulin-III monoclonal  antibody overnight at 4°C. Slides were washed in PBS and biotin-conjugated goat anti-mouse IgG  was applied for 1 h at room temperature. After secondary antibody application, slides were PBS washed and incubated with diluted alkaline phosphatase-conjugated streptavidin  for 1 h. The color reaction was developed for 2 min using the fresh fuchsin substrate system  in the presence of levamisole at a final concentration of 24 µg/ml. All images were acquired using Axiovision (Zeiss) digital imaging software with a Nikon Eclipse E800 microscope.DNA and RNA were extracted from cell pellets using DNeasy and RNeasy kits according to manufacturer's protocol . Complementary DNA was synthesized from 100 ng of DNase-treated RNA using the Transcriptor System  as previously described en passant mutagenesis as described SceI-aphAI and mRFP-I-SceI-aphAI cassette was amplified from pEP-eGFP-in and pEP-mRFP-in, respectively E.coli cells . All clones were confirmed by DNA sequencing using primer sets specific for either ORF63 or ORF70, as well as multiple restriction fragment length polymorphism analyses (RFLP), to ensure the integrity of the genome  or the monomeric red fluorescent protein (mRFP)  using twe genome .Recombinant BAC DNA used for transfection was isolated using the plasmid Midi-prep kit according to the manufacturer's instruction . MeWo cells were transfected with the Lipofectamine 2000 reagent  as described 6) were inoculated with 100 plaque-forming units (pfu) of cell-associated virus. Infected cells were trypsinized at 24, 48, 72 or 96 h post infection (h.p.i.), titrated in 10-fold dilutions, and added to MeWo cell monolayers seeded 24 h before. The number of plaques was determined by indirect immunofluorescence using an anti-VZV antibody exactly as described earlier MeWo cells  according to the manufacturer's instructions Metabolic parameters of infected and uninfected NHNP TLAs were measured every 24 h over the course of the experiments to monitor a cellular development profile and to monitor the metabolic status of the tissues. Glucose consumption was determined using the iStat clinical blood gas analyzer using an EC8


The following information was erroneously excluded from the Funding statement: "This work was also supported in part by the National Institutes of Health under grant U54CA151838 through a pilot project."