text:
CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

Results
The design and validation of sgRNA targeting hPD-1. Previous works have shown that simultaneous use of dual sgRNAs to target an individual gene significantly improved the Cas9-mediated genome targeting efficiency and reduce the site-dependent off-target effect 22 . So we selected 2 pairs of targeted sites on exon 2 (Fig. 1a). All sgRNA expression vectors were co-transfected into HeLa cells separately with Cas9 expression vector to test their efficiency. Blasticidin (5 μ g/ml) and puromycin (1 μ g/ml) were added 24 h after transfection for positive selection. Genomic DNA was isolated from cells harvested 72 h after transfection and screened for the presence of site-specific gene modification by PCR amplification of regions surrounding the target sites as well as T7EN1 cleavage assay. The cleavage bands were all clearly observed (Fig. 1b) and various indels were detected by the sequencing (Supplemental Fig. 1). Mutation sizes ranged from − 86 to + 51 at the efficiency of 61.9% for sg1, 52.6% for sg2, 40% for sg3, 52.6% for sg4, 47.6% for sg (1 + 2) and 38.9% for sg (3 + 4), respectively. These data demonstrated that the selected sgRNAs worked effectively with Cas9 on human genomes. We chose sg (1 + 2) in our following experiments.
Optimizing Cas9/sgRNAs delivery of primary T cells. We next aimed at optimizing conditions for the co-transfection of Cas9/sgRNA expression plasmids on primary T cells via electroporation. For this purpose, we used reporter plasmids pST1374-Cas9-GFP encoding green fluorescent protein (GFP) tagged Cas9 to evaluate the transfection efficacy through the co-electroporation with pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA (1 + 2). Here we referred to several related research based on primary T cells transfection by the Nucleofecter platform and expanded our experimental program beyond the Lonza protocol. To this end, a mixture of 5 μ g of pST1374-Cas9-GFP and 10 μ g of pGL3-U6-hPD-1-sgRNA plasmids was used, and different programs (Y-001, T-007, T-023, X-001, U-014, V-024) were applied. GFP expression was determined 24 h later by fluorescence microscope or flow cytometry. We found that higher transfection efficacy was obtained with program U-014 than with V-024 and T-007 ( Fig. 2a,b, p = 0.0231 and p = 0.0316). The T7EN1 cleavage assay confirmed the PD-1 mutation of T cells (Fig. 2c). Notably, during the following days, significant improved cell viability was observed with program T-007 than with U-014 and V-024 shown by Trypan blue exclusion assay (Fig. 2d, p = 0.0353 and p = 0.0058). Therefore, T-007 was used as the optimal program for the following experiments. The transfection efficacy of different molar ratio of pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA evaluated by GFP expression. (f) Detection of sgRNA1:Cas9-mediated cleavage of hPD-1 by T7EN1 cleavage assay using program T-007 with a molar ratio of 1:4 (pST1374-Cas9-GFP:pGL3-U6-hPD-1-sgRNA). Data shown are mean ± SD of 3 independent experiments. p < 0.05.
In addition, we found significant increase of GFP expression when plasmids were delivered at a molar ratio of 1:4 (pST1374-Cas9-GFP:pGL3-U6-hPD-1-sgRNA) (Fig. 2e). To determine the mutation of PD-1, T7EN1 cleavage assay was applied and the gene knockout efficacy was proved (Fig. 2f). These results indicated that human PBMC-derived T cells were efficiently modified by sgRNA:Cas 9 system through the electroporation of plasmids.

Cas9-mediated efficient PD-1 KO in primary T cells of patients.
To test whether the Cas9-mediated gene knockout was as efficient in patients as in healthy donors, primary T cells from two late stage cancer patients and healthy donors were transfected. Freshly isolated PBMC were activated for 3 d. A total of 10 μ g of pST1374-Cas9-GFP and 20 μ g of pGL3-U6-hPD-1-sgRNA plasmids were used for each reaction. The GFP expression was evaluated by fluorescence microscope 24 h after electroporation. We found that PBMC-derived T cells from patients (Donor 1 and Donor 2) (Fig. 3a) achieved less transfection efficacy comparing with T cells from healthy donor (Donor 3) (Fig. 3a). Over 85% viable cells were observed in all three donors evaluated by Trypan blue exclusion assay (Table 1). To determine the subsets of T cells, surface expression of CD3, CD4 and  CD8 were calculated on day 7 of culture. The results showed that over 85% of cells were CD3 + T cells and over 80% of CD3 + T cells were CD8 + cells in all three donors (Table 1). PCR results showed there harbored large fragment deletion on samples from healthy donor (H2), also indicating the higher efficacy of gene disruption than the other two donors (G2 and Z2). The subsequent T7EN1 assay and sequencing further confirmed that the mutation of PD-1 was successfully achieved in all three donors (Fig. 3b). Further characterization of the cleavage by sequencing showed, different indels were detected in all the donors with various mutation sizes, the efficacy varied among donors (10.71% in Donor 1, 14.81% in Donor 2, 66.67% in Donor 3) (Fig. 3c). These results demonstrated that the efficient disruption of gene PD-1 is achieved by sgRNA:Cas9 system although the efficacy of the gene modifications varied among different individuals.
The proliferation of gene edited primary T cells and the sustained knockout of PD-1 during the prolonged culture conditions. As adoptive T cell therapies require relatively long culture of T cells in vitro, we are curious how long the down regulation of PD-1 lasted by our gene disruption method. To this end, we first assessed the surface expression of PD-1 48 h post transfection. Here we depicted a representative out of three experiments yielding similar results. The percentage of PD-1 + T cells was 2.96% on control T cells and was 1.37% on the sgRNA:Cas9 modified T cells (Fig. 4a), though the baseline expression of PD-1 was relatively low. Then, we assessed the capacity of sgRNA:Cas9-treated T cells to proliferate in vitro upon stimulation with IL-2 by counting the total cell numbers. Over a period of 21 d, we found the proliferation of primary T cells of all these donors was not significantly affected by the disruption of PD-1 during the prolonged culture period, ranging from 2 × 10 7 cells on day 7 to 20 × 10 7 on day 21, and the fold increase was 1.68 ± 0.03, 7.57 ± 0.09 and 10.64 ± 0.27 on day 7 (p = 0.8816), day 14 (p = 0.8557) and day 21 (p = 0.7705), respectively (Fig. 4b,c). T cell clones were observed around day 7 and grew largely during the following days, indicating good proliferation and activation of the T cells (Fig. 4d). Meanwhile, T cells from a healthy donor were transfected and stimulated by autologous DCs loaded with immunogenic peptides for another two weeks. The surface expression of PD-1 of CD3 + T cells increased from 2.44% at the baseline to 4.48% of the PD-1 KO group while it increased from 5.92% to 15.9% in control group (Fig. 4e). To further mimic the in vivo environment when T cells encounter with whole tumor antigen, we co-cultured the gene modified T cells with irradiated PD-L1-high tumor cells. We observed that only 3.4% of sgRNA:Cas9 modified T cells express PD-1 while 17.5% of control T cells express PD-1 by the stimulation of whole tumor antigen (Fig. 4f). Taken together, these data represented that the proliferation capability was not affected by the disruption of PD-1 on primary T cells and that the efficient disruption of PD-1 sustained during the prolonged culture with the stimulation of tumor antigens.

The characterization of the cultured T cells by sgRNA:Cas9 mediated knock out of PD-1.
To characterize the cultured T cells with PD-1 disruption, we harvested T cells on day 21 post transfection. The T cell subsets were determined by the expression of CD4, CD8 and characterized with memory or activation markers, including CD28, CD27, CD69 and HLA-DR. The results showed, the PD-1 knock out T cells did not exhibit any significant change on the expression of CD4 (7.43 ± 2.78% on control T cells vs 13.36 ± 6% on sgRNAhPD-1 T cells, p = 0.1957) or CD8 (89.10 ± 4.5% on control T cells vs 81.87 ± 7.82% on sgRNAhPD-1 T cells, p = 0.2367) during the extended in vitro culture after gene editing (Fig. 5a). Although the decrease of CD4 + CD25 + cells on PD-1 KO T cells (4.48% vs. 2.42%) was observed in one of the donor (Fig. 5b), there was no statistical significance, p = 0.4604. It is noteworthy that, we did not find any significant change of the memory markers ( Fig. 5c), including central memory CD45RO + CD62L + T cell (26.87 ± 3.48% vs 31.53 ± 3.1%, p = 0.1583), effector memory CD45RO + CD62L − T cell (14.76 ± 5.44% vs 15.77 ± 2.58%, p = 0.7858) and naïve T cell (44.1 ± 5.79% vs 42.1 ± 8.08%, p = 0.7451). No difference of the activation marker CD28, CD27, CD69 and HLA-DR on CD3 + T cells was detected between the sgRNAhPD-1 T cells and control T cells (p > 0.01) (Fig. 5d). These results indicated that the CD4 or CD8 subsets constitution or the memory and activation status of the T cells is stable with sgRNA:Cas9-mediated PD-1 disruption.

Discussion
The effective activation of the tumor reactive T cells and the suppression of checkpoint inhibitor has long been the key problem of immunotherapy. Recently, the utilization of checkpoint blockade targeting the PD-1/ PD-L1 pathway has shown remarkable antitumor responses in patients with advanced melanoma, lung cancer and against other cancers with durable clinical responses [23][24][25][26] . T cells activated in the absence of PD-L1/PD-1 co-stimulation are functional activated, exhibiting increased proliferation by the stimulation of DCs or tumors and produce higher levels of Th-1 cytokines, in particular IFN-γ , IL-2 and TNF-α and enhanced lytic activities 27 . However, most of these studies have been performed using either blocking antibodies or RNA interference with Adoptive cell therapy using autologous gene editing T cells such as TCR-T or CAR-T has emerged as promising approach for the treatment of cancers 30 . There is demonstration that blockade of the PD-1 immunosuppressive pathway using an anti-PD-1 antibody significantly enhance the anti-tumor efficacy of genetically modified T cells expressing a chimeric antigen receptor (CAR) 31 . Despite the dramatic benefit achieved by these strategies, it has to be noted that in one hand, sustained expansion of PD-1-expressing CTLs by the stimulation of tumor antigens in vitro and in vivo may require continuous treatment with anti-PD-1 antibody which is costly, on the other hand, the long term systematic administration of the blocking antibody carries the risk of breaking immune tolerance may cause immune attack of normal tissues. In addition, RNA interference with siRNAs on T cell may be temporary and less efficient.
In our study, we described, for the first time, a new approach of inhibiting PD-1/PD-L1 co-stimulation by directly disrupting genome PD-1 expression on human primary T cells through the Cas9:sgRNA gene knock out system. This was achieved by electro-co-transfer of two DNA plasmids into cultured human T cells. A major obstacle of editing primary T cell is the low transfection efficacy. Here, we made use of nucleofection to achieve higher transfection efficacy and better cell viability. This non-viral mediated gene disruption method has the advantage of clinical application at affordable costs 32 . To optimize the established protocol for co-transfection of primary T cell, by using a GFP reporting plasmid, we observed significantly improved cell viability with program T-007 than with U-014 and V-024 which are mostly used in the studies of T cell editing 33 . In addition, the ratio of co-transfection of two plasmid were vital for the efficient gene disruption of sgRNA:Cas 9 system. Interestingly, our approach to disrupt PD-1 expression on human T cells was successfully utilized on several cancer patients and healthy donors as the requirement of T cell editing for adoptive transfer of patient's autologous lymphocytes or in some cases allogenetic lymphocytes of healthy donors. We found that PBMC-derived T cells from patients achieved less transfection efficacy comparing with T cells from healthy donors. Those functional impaired T cells may account for the low sensitivity to electroporation. The phenomenon inspired us to testify this approach firstly in the allogenetic adoptive therapy of cancer treatment using PBMC from healthy donors. Moreover, we observed for over 3 w after PD-1 gene disruption, and found no significant differences in the cell expansion rate between sgRNA:Cas9-treated cells and control cells, which encouraged us to further apply this approach into clinical application in future. In addition, the activation induced up-regulation of PD-1 was disrupted through the in vitro expansion stimulated by peptides pulsed DCs or irradiated whole tumor cells. As we know that the most popular schedule for T cell in vitro culture would not surpass 3 ~ 4 w.
Previous studies already demonstrated that blockade of PD-1/PDL1 by mAb improved IFN-γ production as well as cytotoxicity both in vitro and in vivo 29,31,34 . Here we also demonstrated that these sgRNA hPD-1:Cas9 modified primary T cells from healthy donors or late stage cancer patients exhibited enhanced IFN-γ production by stimulating with the related peptide antigens and at the same time we found improved tumor cells lysis by the disruption of PD-1 which may due to the reversed immune resistance mediated by PD-1/PDL1 interaction. IFN-γ is one of Th1 cytokines which mediated cellular immune response and activate cytotoxic T cells, indirectly regulate tumors lysis by several mechanisms 35 . Therefore, we believe in our case, IFN-γ activates cytotoxicity indirectly. Additionally, in our system using Cas9:sgRNA mediated gene editing of T cells from both patients and healthy donors, improved cytotoxicity on tumor cell lines was clarified on two PD-L1 positive target cell lines and further testified by induction of PD-L1 expression on the target cell. The expression of its receptor PD-L1 should be taken into consideration for good outcome of utilizing PD-1/PD-L1 inhibiting strategy 36 .
Nevertheless, our study did not precisely focus on a specific antigen and our exploration was nor clearly clarified in an antigen dependent manner. We are currently explore PD-1 disruption of T cells by generating CTL targeting on specific tumor antigen, as previous studies reported that sustained expansion of PD-1-expressing CTLs may require continuous treatment to interrupt PD-1/PD-L1 co-stimulation 27 . In addition, as it is difficult to obtain large number of PD-1 high expression T cells from periphery lymphocytes, the difference of the functional analyzes between our "PD-1 KO cells" and "control cells" need further study. We sought to use the approach in the following research by changing the model into PD-1 high expression tumor infiltrating lymphocytes in solid tumors or tumor associated lymphocytes in ascites or pleural effusion of cancer patients, as they expose intensively to tumor antigens and acquired adaptive immune resistance that mediates resistance to immunotherapies 37 .
In conclusion, we established an sgRNA:Cas9-basd effective gene disruption method for the highly efficient disruption of PD-1 on primary human T cells. This easy handling technique has great potential to achieve nice effect. And this electroporation mediated approach provides an alternative to labor-intensive and time-consuming viral-mediated gene transfer methods. Therefore, our gene editing method might be suited for both research and clinical applications and we are confident that it will be beneficial to cancer adoptive cell transfer treatments using tumor-specific lymphocytes in the near future.

Materials and Methods
Ethics statement. All the experimental methods were carried out in accordance with the approved guidelines. The blood collection procedure was carried out in accordance with the guidelines verified and approved by the Ethics Committee of Drum Tower Hospital. All donors signed an informed consent for scientific research statement.
Plasmid expression vectors. The Cas9 expression construct pST1374-Cas9-N-NLS-Flag-linker (Addgene 44758) was modified by adding an EGFP sequence to its C terminal. Oligos (Supplementary Table 1) for generation of sgRNA expression plasmids were annealed and cloned into the BsaI sites of pGL3-U6 sgRNA-PGK-Puro vector. The pGL3-dual U6-sgRNAs-PGK-Puro vector was produced based on pGL3-U6 sgRNA-PGK-Puro vector (Addgene 51133), which was modified by removal the small fragment between two BsaI sites and replaced with a ccdb suicide gene. The BsaI sites were also substituted for Esp3I sites. Taken the origin U6 promoter and sgRNA scaffold on pGL3-U6 sgRNA-PGK-Puro as the 1 st sgRNA promoter and the 2 nd sgRNA structure, primers containing pairs of 20 bp targeted sequences (Supplementary Table 2) were used to PCR-amplify the 1 st sgRNA scaffold and the 2 nd U6 promoter from a pUC57-sgRNA-U6 plasmid. The PCR products were then digested with Esp3I and the ccdb gene was replaced, forming the tandem dual sgRNA expression vectors.
Preparation of primary human PBMCs. Apheresis specimens were collected from stage III/IV cancer patients or healthy donors. PBMCs were isolated by centrifugation on a Ficoll density gradient and suspended in AIM-V medium (Gibico, USA). Cells were frozen in 90% FBS serum (Gibico, USA), and 10% dimethyl sulfoxide (Sigma, USA). All PBMCs were used for experiments or stored in a secure liquid nitrogen freezer until use.
T cell activation and electroporation. PBMC were cultured by adherence for 1 ~ 2 h and non-adherent cells were moved and suspended in AIM-V medium supplemented with 1000 U/ml IFN-γ on day 1 and 50ng/ml OKT-3 (eBioscienc, USA) and 300 U/ml of human recombinant IL-2 (eBioscienc, USA) on day 2 for 2 ~ 3 d. Cells were transfected with the intended plasmids by Nucleo-fector 2B (Lonza, Germany) using the Amaxa Human T cells Nucleofector Kit, VPA-1002 (Lonza, Germany). 5 ~ 10 × 10 6 cells were washed twice with DPBS by centrifuging at 800 rpm for 5 m and resuspended in 100 μ l transfection buffer and then transferred into the electroporation cuvette. Program T-007 was selected for both high transfection and high efficiency. After electroporation, cells were resuspended in 500 μ l pre-warmed AIM-V medium containing 10% FBS and transferred into 6-well cell plate and incubated at 37 °C in 5% CO 2 . The transfection efficiency was evaluated by the fluorescent counts 24 h after electroporation. Cells culture medium was half replaced by fresh complete medium containing IL-2 (100 ~ 300 U/ml) every 2 ~ 3 d. Cell viability was evaluated by flow cytometry 24 h after electroporation. T7EN1 cleavage assay and sequencing. Cells were harvested and digested with 100 μ g/ml Proteinase K in lysis buffer (10 μ M Tris-HCl, 0.4 M NaCl, 2 μ M EDTA and 1% SDS). Genomic DNA was extracted by phenol-chloroform and alcohol precipitation. The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50). Purified PCR product was denatured and re-annealed in NEBuffer 2 (NEB) using a thermocycler (ABI, Veriti9902). Hybridized PCR products were digested with T7EN1 (NEB, M0302L) for 30 m and separated by 2% agarose gel. Primers for PCR are listed in Supplementary Table  3. The purified PCR products were ligated with pMD19T vector (Takara, 6013) using DNA ligation Kit Ver. 2.1 (Takara, 6022). Ligation products were used for transformation and about 20 ~ 30 colonies per kind are sequenced by using universal primer M13F.
In vitro generation of autologous DC. DCs were generated from monocytes enriched by adherence for 1 ~ 2 h, and cultured in AIM-V medium containing 10% FBS together with human GM-CSF (500 U/ml, Peprotech) and IL-4 (500 U/ml, Peprotech) to obtain immature DCs. To obtain mature DCs (mDCs), fresh complete medium containing TNF-α (500 U/ml, Peprotech), IFN-α (500 U/ml, Peprotech) and PGE2 (50 ng/ml, Peprotech) was added to the culture on day 5. The culture was continued for an additional 2 d. On day 7, all DCs were harvested to be frozen or used for experiments. As described previously, these DCs possess the ability to present peptide antigen and express CD80, CD86, HLA-DR and CD11c.

In vitro expansion of PD-1 KO T cells. Control T cells or PD-1 KO T-cells post transfection were cultured
in AIM-V medium supplemented with IL-2 (300 U/ml, Peprotech) and half replaced by fresh complete medium containing IL-2 every 2 ~ 3 d until analyzes. For antigen stimulated T cells. Mature DCs were pulsed by peptide (25 μ g/mL) for 2 ~ 3 h at 37°C, washed with pre-warmed PBS and then incubated with control T cells or PD-1 KO T-cells at a ratio of 1:10 in complete AIM-V medium supplemented with IL-2 (100 U/ml, Peprotech) in 6-well-plates (5 × 10 6 cells/well) on day 7 post electroporation. IL-7 and IL-15 (5 ng/mL, Peprotech) were added with fresh medium. For re-stimulation, autologous DCs were pulsed with peptide (25 μ g/mL) for 2 h and added to the cultured cells for another 7 d. Fresh complete medium was added containing cytokines every 2 to 3 d until use for experiments.
ELISPOT assay. IFN-γ ELISPOT kit (Dakewei, China) was used to determine the frequency of cytokine-expressing T cells after overnight activation with peptides. Briefly, T cells (10 5 per well) and peptides (50 μ g/ml) were added to duplicate wells and DCs were added at ratio (DC:T) of 1:5 ~ 1:10 for 18 ~ 20 h. The plates were washed before the addition of the diluted detection antibody (1:100 dilution) and then incubated for 1 h in 37 °C. After washing the plates, streptavidin-AP (1:100 dilution) was added and incubated at 37 °C for another 1 h. AEC solution mix was then added to each well, and the plates were left in the dark for about 15 ~ 25 m at room temperature before deionized water was added to stop development. Plates were scanned by Elispot CTL Reader (Cell Technology Inc, Columbia, MD) and the results were analyzed with Elispot software (AID, Strassberg, Germany). Cytotoxicity assay. Transduced T cells were tested for lytic activities by CFSE/PI labeling cytotoxicity assay.
Target tumor cells were labeled with 4 μ M CFSE (Carboxyfluorescein succinimidyl ester) (Invitrogen) for 10 m at 37 °C in PBS. Labeling was stopped by adding 10 fold volume of PBS and extensively washed in PBS before seeding into the 24-well plates. CFSE-labeled cells were then incubated with T cells by different effector to target ratio for 6 ~ 16 h. Propidium iodide (PI) (Sigma) was added to determine the ratio of cell death. Samples were analyzed by flow cytometry. Statistical analysis. Graphpad Prism 5.0 (Graphpad software, San Diego, CA) was used for all statistical analysis. The mean ± S.E.M. was determined for each treatment group in the individual experiments. And the one-tailed Student t-test was used to determine the significances between treatment and control group. P-values < 0.05 were significant.


extfieldsofstudy: Biology Chemistry

s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
A New Strategy for Mitigation of the Allergenic Activity of Ovomucoid in Hen Eggs and Beta-Lactoglobulin in Cow Milk

Recently, public interest in food allergies has been increasing because of increasing prevalence of these allergies among children. In Japan, hen egg and cow milk allergies account for nearly 50% of all cases of childhood food allergies. Patients with hen egg and cow milk allergies should avoid these foods and products that contain them until they have outgrown their allergies. However, regarding nutrition, quality of diet, and hyposensitization, it is significantly important for these patients to ingest hypoallergenic hen eggs, cow milk, and products that contain them. We previously demonstrated the mitigation of the allergenic activity of beta-lactoglobulin in cow milk and ovomucoid in hen egg by electrolysis. In this review, we discuss this new strategy for producing hypoallergenic hen eggs and cow milk.

white has been considered the most important source of allergens, with the ovomucoid (OMC; WHO/IUIS allergen name: Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), and lysozyme C (Gal d 4) being the major allergens in it, but IgE-binding allergens have also been identified in the yolk. The characteristics of these four major egg white allergens are shown in Table 1. OMC and ovotransferrin are the most frequent allergens though it may depend on the reports.
Cow milk contains around 30-35 g of proteins per liter and includes more than 25 different proteins but only some of them are known to be allergenic. Table 2 provides the characteristics of cow milk allergens. Cow milk proteins consist of the coagulum containing the casein proteins and the lactoserum (whey proteins) representing 80% and 20% of the total milk proteins, respectively [3]. The casein fraction (Bos d 8) consists of four proteins that account for different percentages of the whole fraction: alphaS1-casein (Bos d 9), alphaS2casein (Bos d 10), beta-casein (Bos d 11), and kappa-casein (Bos d 12), with alphaS1-casein being the most important allergen of the casein fraction [4]. Allergens in the whey fraction are alpha-lactalbumin (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5), immunoglobulins (Bos

Introduction
Food allergies continue to be an important human health problem. Recently, cases of food allergies have been increasing, with 5%-10% of infants and 1%-3% of school-age children in Japan affected by food allergies [1]. Hen eggs are most commonly responsible for food allergies, followed by cow milk. Patients with these food allergies develop adverse reactions, like urticaria, angioedema, wheezing, gastrointestinal symptoms, or anaphylactic shock, after ingestion of hen eggs, cow milk, or products that contain them.

Diagnosis of hen egg and cow milk allergy and effectiveness of the skin prick test
Conclusive diagnosis is crucial in patients with suspected food allergies. Double-blind, placebo-controlled food challenges are still the gold standard for diagnosing food allergies; however, these are time consuming, expensive, and troublesome for patients and involve risks of severe systemic reactions [7].
Several diagnostic tests have been developed to predict outcomes of oral food challenges. Analysis of food-specific serum IgE levels [8,9], the skin prick test (SPT) [10][11][12], and the atopy patch test [13] may be useful tools for diagnosing food allergies. However, the food challenge is still a crucial tool for definitively diagnosing food allergies, as analysis of food-specific serum IgE levels and the SPT do not currently render oral food challenges unnecessary in most cases [14]. The SPT is an important first-line procedure for evaluating food allergies, as it is quick and relatively inexpensive. Previously, we reported that measuring wheal sizes in the SPT and calculating the skin index (SI) could help diagnose many food allergies (e.g., to hen eggs, cow milk, wheat, and peanuts) [15]. Moreover, we performed the SPT in 126 children suspected to have CMA and 76 had positive oral provocation test results. We performed a logistic regression analysis to evaluate whether wheal diameters or SIs could predict CMA in these 126 patients. We found that wheal diameters and SIs could predict positive oral provocation test results. While the expected probabilities of having a positive oral provocation test result were ≥ 0.5, the cut-off values for wheal diameters and SIs were ≥ 8 mm (79.5% sensitivity and 76.7% specificity) and ≥ 1.0 (77.3% sensitivity and 78.9% specificity), respectively.
Hill et al. [11] and Sporik et al. [16] showed that a 6 mm wheal diameter in children < 2 years of age, or 8 mm in children > 2 years of age, indicates 100% specificity of a clinical reaction. Saarinen et al. [17] reported 98% specificity and 92% positive predictive value (PPV) for > 8 mm wheal diameter. Verstege et al. [12] reported 95% PPV for a 12.5-mm wheal diameter when pricked with fresh milk. Calvani et al. [18] reported 95% PPV for a 15 mm wheal diameter in infants; this evidence supports results of our study, which shows that a 15 mm wheal diameter had 90% PPV.

Treatment of hen egg and cow milk allergy
Patients with hen egg allergy (HEA) or cow milk allergy (CMA) should avoid these foods or products containing them until they outgrow HEA or CMA. However, it has been recently reported that oral immunotherapy by ingestion of these foods was able to achieve effective desensitization to these food allergies [19]. The egg white has a higher allergenic content than the yolk of the hen egg. Moreover, because raw hen eggs have greater allergenic content compared to cooked eggs, patients with HEA are likely to develop adverse reactions after ingestion of raw egg whites in the oral provocation test (OPT). Many patients with HEA can ingest the cooked yolk, and some can even ingest the cooked egg white in small amounts. We should therefore assess their upper dose limit in the oral hen-egg provocation test. It is certainly important for daily nutrition and quality of diet of patients to be able to ingest a small amount of cooked egg.
Extensively hydrolyzed formulas (ehCMF) have been recommended as milk substitutes for infants with CMA. However, together with their unpleasant taste, these formulas present nutritional issues, such as growth reduction [20], decreased plasma protein concentrations and iron-binding capacity, and unbalanced plasma and urinary concentrations of some amino acids [20,21]. Furthermore, animals given hydrolysate formulas exhibited enhanced trypsin and chymotrypsin proteolytic activities in the intestine, higher cytochrome levels in the liver, and disrupted glucocorticoid metabolism [22]. Therefore, mitigation of the allergic action of cow milk, independent of the use of proteolytic enzymes, may be required. Partially hydrolyzed cow milk formulas (phCMFs) present therapeutic effects in infants with mild to moderate atopic dermatitis [23]. Although phCMFs are not considered substitute formulas for infants with CMA, we recently reported that 40 of 53 (75%) children with CMA could ingest phCMF [24] without any adverse reactions. Additionally, most children presenting a >6-mm wheal diameter with phCMF could complete the OPT with phCMF [24]. Because phCMF includes larger molecular weight proteins compared to ehCMF [24], which do not pass through an ultra-filtration membrane, phCMF has higher allergenicity compared to ehCMF. Since phCMF is expected to retain more cow-milk antigens compared to ehCMF, early and longterm administration of phCMF may induce immunologic tolerance to cow milk antigens in children with CMA [25,26]. Extensive hydrolysis of cow milk generates considerable quantities of free amino acids, which causes bitterness. Since phCMF has been reported to taste better than ehCMF [27], phCMF may be more beneficial than ehCMF as a nutritional ingredient for drinks and food.

Beta-lactoglobulin
Beta-lactoglobulin (BLG), which is a prominent S-S protein allergen, is the main protein in whey, and has no counterpart in human breast milk. BLG is composed of nine beta-strands (beta-strand A-I) and one alpha helix; it is assumed that intermolecular hydrogen bonds between beta-strand-I main chains and intermolecular salt bridges between side chains of the loop (beta-strand-A and -B) play important roles in dimer formation [28]. The allergenic peptide 41V-60K contains beta-strand-B (41V-50P); the other allergenic peptide, 149L-162I, includes part of beta-strand-I (146H-151F). As B-cell epitopes are mostly conformational and dependent on the threedimensional structure of antigens, heat denaturation of BLG has been studied to modulate its immunogenicity and allergenicity. Although decrease in IgE-binding ability of BLG has been demonstrated after heat treatment to some degree [29], heat-denatured BLG induces a more intensive local immunologic reaction in the gastrointestinal mucosa compared to native BLG [30].
The antigenicity of BLG has been reported to depend on its intramolecular S-S bonds, and can be reduced by thioredoxin through its catalytically active S-S groups, which causes lower allergenicity and enhanced digestibility [31].

Ovomucoid
The allergen protein content in hen eggs is mainly included in the egg white. Ovomucoid (OMC) has been identified as the most prominent allergen causing HEA and contains S-S bonds that may cause its allergic action [32,33]. OMC, composed of 186 amino acids,  [34]. Gal.d1.1 contains an α-helix and three β-sheets. Each domain has three intramolecular S-S bonds, but no intermolecular S-S bond [35]. OMC is highly resistant to proteolytic enzymes and heat [36] and exhibits high allergic activity due to this structural feature.

Electrolyzed beta-lactoglobulin
Although reduction of S-S bonds was expected during electrolysis on the cathode side, only short-term modulation of the redox potential and a low voltage difference was observed for whey proteins with a carbon electrode [37,38]. However, carbon electrodes are not approved for the modulation of food in Japan.
We reported that the allergic activity of BLG was markedly mitigated by a much higher voltage difference between platinum electrodes [39]. Briefly, we electrolyzed BLG as follows (Figure 1 in Kido et al. [40]). Heat-tight, 30 mm bore glass tubes were used in an H-shaped electrolysis cell, connected with two tubes of 300 mm in length and one tube of 40 mm in length. The electrolysis was carried out using 1% BLG containing 1% sodium chloride for 30 minutes with a voltage difference of 90 V, 0.23 A (30 mA/cm 2 ). The temperature of the electrolyte in both glass tubes was kept in the range of 12-18°C and the temperature next to the electrodes ranged from 40°C to 50°C during the electrolysis. Each BLG solution was then filtered through a 0.45 mm filter unit.
In patients with CMA, the wheal size for BLG on the cathode side (cBLG) was markedly reduced in the SPT compared with untreated BLG (uBLG) or BLG on the anode side (aBLG), except in one patient with a serious systemic anaphylactic reaction to milk. The percent decrease in the wheal reaction with cBLG compared to uBLG (mean 71% ± 28%) was similar to that observed on comparison of the ehCMF with regular milk (mean 79% ± 18%), whereas the wheal size with aBLG was not affected significantly compared with uBLG [39]. Because the quantity of SH groups of BLG was decreased almost equally on both anode and cathode sides, the modulation of S-S bonds of BLG did not appear fundamentally associated with mitigation of its allergic action.
It has been postulated that the B-cell epitope of a protein with a rigid globular structure is localized on the molecular surface. Selo et al. [41] identified three peptide fragments as allergenic sites on the BLG molecule after tryptic digestion, i.e., residues 41V-60K, 102Y-124R, and 149L-162I, all of which are exposed outside its rigid three-dimensional structure [42]. In our study, trypsin treatment of uBLG and aBLG clearly identified the two fragments, 41V-60K and 149L-162I, whereas no corresponding tryptic fragment was recovered for cBLG or apparent monomeric forms of BLG derived from the untreated or anode spots. The uBLG was detected as a single peak of a dimeric form of BLG in the gel filtration study [39], suggesting that native BLG is present in milk as a dimeric form. Allergic action was observed for uBLG and aBLG, and two antigenic fragments were distinctly separated in gels containing dimeric forms of uBLG and aBLG. Therefore, it seems likely that the dimeric form is highly involved in the allergic action of BLG, and the allergic action of the dimeric form of BLG is closely associated with two peptide fragments, 41V-60K and 149L-162I, on its surface [39]. This is of special interest, as the dimer and oligomers of cBLG showed similar mass-spectrometric patterns compared to monomeric BLG [39]. It is crucial to mitigate the sensitization potential of these modified proteins in nutritional formulas for infants at high risk of developing food sensitivity.
The allergenic peptide 41V-60K contains beta-strand-B (41V-50P), and the other allergenic peptide, 149L-162I, includes a part of beta strand-I (146H-151F). An SDS-PAGE study with complete reduction showed 35-and 16-kDa bands for cBLG, whereas a single 18-kDa band was seen for both uBLG and aBLG [39]. Alternatively, the dimeric structure of cBLG might be different from that of native BLG, causing conformational modulation with dislocation of allergenic peptides from the surface of BLG. Fifteen of 162 amino acids in the sequence of BLG are lysine residues. Lysine is an essential amino acid for the maintenance of the antigenic structure of BLG [43]. There were slightly fewer lysine residues on the cathode side than on the anode side. Various commercial BLG products are reported to be lactosylated to some degree [44], and reducing sugars like lactose favor the Maillard reaction via a Schiff base, between the aldehyde of the glucose moiety of lactose and an amino group of a protein, under conditions of heat and optimum pH [45]. Because a lactose-binding site of BLG was identified as being a lysine residue [44], the decrease in lysine residues may be due to the Maillard reaction during electrolysis.
Mitigation of the allergic action of cow milk, independent of hydrolyzation, may be beneficial, as there are some concerns about nutritional issues regarding hydrolysate formulas [20][21][22]. Additionally, the taste of whey, following electrolysis on the cathode side, was not altered. Therefore, our method may offer a new and more advantageous strategy of preparing hypoallergenic formulas for patients with CMA.

Electrolyzed ovomucoid
There have been some reports of efforts to obtain hypoallergenic OMC. Cooke and Sampson [46] reported that the binding force of OMC to IgE antibodies decreased by approximately 28% when its intramolecular S-S bonds were cleaved by dithiothreitol (DDT), and this was particularly demonstrated in infants with hen egg allergy. Kato et al. [47] reported that the allergenic capacity of OMC was diminished when OMC was heated with wheat protein and was aggregated. This phenomenon was attributed to formation of intermolecular S-S bonds between the OMC and wheat protein, and the aggregation of OMC. When the solid component concentration of egg protein was 0.2%-0.3%, pH 10-11.5, and OMC was heated to > 80°C, OMC was denatured and lost its high allergenic capacity [48]. Moreover, when OMC was treated at high pressure (100-400 MPa), the binding force between it and IgE antibodies decreased. Thus, tyrosine residues of OMC moved from the molecular entrail to the molecular surface.
We also showed that the allergenic capacity of OMC was attenuated after energization [40]. The electrolysis was carried out in the same way using 1% BLG containing 1% sodium chloride for 30 minutes with a voltage difference of 90 V, 0.23 A (30 mA/cm 2 ). The wheal diameter for OMC on the polar opposites was reduced in the SPT compared with that of untreated OMC. In 21 patients with hen egg allergy, percentages of the wheal reaction with OMC on the cathode (cOMC) and anode (aOMC) sides, compared to untreated OMC (uOMC), were 77.6% ± 28.8% and 82.6% ± 33.8% (P = 0.004, P = 0.030), respectively [40].
This phenomenon of mitigated allergenicity for OMC was evidently demonstrated in the cOMC and the quantity of SH groups in cOMC significantly increased. Some of nine S-S bonds in an OMC molecule were expected to be cleaved, which is in accordance with Cooke and Sampson's report [8] that the binding ability between OMC and IgE decreased when S-S bonds were cleaved upon addition of DDT. Our study suggests that an intramolecular S-S bond of OMC was cleaved by electric energy without use of a reducing agent such as DDT, and this cleavage contributed to decreased allergenic capacity [40]. We performed 2D electrophoresis with an irreducible solvent for uOMC, cOMC, and aOMC, with some extremely diverse spots being obtained (Figure 4 in Kido et al. [40]). We cut these relatively large spots out of the three respective gels, treated these proteins with trypsin, and then performed mass spectrometry for the peptide fragments of each gel. Moreover, we calculated the theoretical molecular weight of OMC and compared the theoretical value to the actual one obtained using mass spectrometry. In cOMC, a new formation of S-S bonds in 15E-24K, 57E-82K and 122R-128R, or 15E-24K, and 165C-185K patterns were expected. In aOMC, a new formation of S-S bonds in 24M-14K, 130E-159K and 57E-63K, 165C-185K, and 18D-24K, or 90A-121K and 18D-24K patterns were expected. Therefore, the S-S bond regions in cOMC and aOMC were likely to have changed variously due to reactions other than reduction, and these changes were considered to contribute to the formation of hypoallergenic OMC.

Conclusion
In Japan, HEA and CMA account for nearly 50% of all cases of childhood food allergies. Although patients with HEA or CMA should avoid hen eggs, cow milk, or products that contain them, until they can outgrow HEA or CMA, it is significantly important for them to ingest hypoallergenic hen eggs, cow milk, or products containing them. An understanding of the mitigation of the allergenic activity of BLG and OMC by electrolysis may allow for its potential use in development of hypoallergenic hen egg or cow milk products or cooking methods.


extfieldsofstudy: Biology Medicine

text:
Sexual reproduction in the Caribbean coral genus Isophyllia (Scleractinia: Mussidae)

The sexual pattern, reproductive mode, and timing of reproduction of Isophyllia sinuosa and Isophyllia rigida, two Caribbean Mussids, were assessed by histological analysis of specimens collected monthly during 2000–2001. Both species are simultaneous hermaphroditic brooders characterized by a single annual gametogenetic cycle. Spermatocytes and oocytes of different stages were found to develop within the same mesentery indicating sequential maturation for extended planulation. Oogenesis took place during May through April in I. sinuosa and from August through June in I. rigida. Oocytes began development 7–8 months prior to spermaries but both sexes matured simultaneously. Zooxanthellate planulae were observed in I. sinuosa during April and in I. rigida from June through September. Higher polyp and mesenterial fecundity were found in I. rigida compared to I. sinuosa. Larger oocyte sizes were found in I. sinuosa than in I. rigida, however larger planula sizes were found in I. rigida. Hermaphroditism is the exclusive sexual pattern within the Mussidae while brooding has been documented within the related genera Mussa, Scolymia and Mycetophyllia. This study represents the first description of the sexual characteristics of I. rigida and provides an updated description of I. sinuosa.

INTRODUCTION
Reproduction in corals consists of a sequence of events which include: gametogenesis, spawning (broadcasters), fertilization, embryogenesis, planulation (brooders), dispersal, settlement and recruitment (Harrison & Wallace, 1990). The success of the reproductive effort is determined largely by the timing, duration, frequency and intensity of the aforementioned events (Babcock et al., 1986). In corals, sexual pattern, mode of reproduction, fertilization, larval dispersal, recruitment and survivorship are key components in determining evolutionary fitness (Szmant, 1986;Edmunds, 2005;Vermeij, 2006;Weil, Croquer & Urreiztieta, 2009;Pinzon & Weil, 2011) which is defined as the product of sexual output (fecundity) and survivorship (Metz, Nisbet & Geritz, 1992). Consequently, the ability of coral species to adapt to modern-day environmental pressures depends greatly on the ability of species to reproduce effectively.
The reproductive characteristics of some scleractinian groups have been more thoroughly studied than others; however, little is known about the reproductive patterns This study characterizes the reproductive biology of I. rigida and I. sinuosa in terms of sexual pattern, mode of development, gametogenic cycles, and fecundity. These fundamental aspects of the physiology of this taxa are understudied. Knowledge of the reproductive biology and ecology of coral species is important for the interpretation of their population and ecological dynamics, their patterns/potential for dispersal, and their local and geographical distribution. The threats currently faced by coral reefs and the ongoing global effort to understand why corals are dying highlight the need to expand our understanding of basic coral physiology.

MATERIALS AND METHODS
Sampling for this study was carried out at La Parguera Natural Reserve, off the southwest coast of Puerto Rico (Fig. 2). This complex reef environment is among the many regions experiencing deterioration by anthropogenic and environmental climate influences at local and global scales. Coral reefs in La Parguera are important local economic drivers, supporting artisanal and recreational fishing, tourism, recreational activities and also protect coastal settlements, seagrass communities and other wetland habitats from the effects of hurricanes and coastal erosion (Ballantine et al., 2008).
At least five unique sample cores were collected monthly for 14 months between March 2000 and May 2001 (Fig. 3A). A total of 89 samples of each species were collected. Colonies were selected by searching in a zig-zag pattern over the distributional range of both species (5-18 m Sample cores were placed in Zenker Formalin (Helly's solution) for 24 h, rinsed and then decalcified in 10% HCl solution. Tissues were then cleaned and placed in plastic tissue holders. Preserved samples were sequentially dehydrated in the rotary tissue processor under 70 and 95%, ethanol, Tissue Dry, and xylene solution (Tissue Clear III). Samples were embedded into Paraplast blocks then sectioned using a rotary microtome. The 8-10 strip sections (7-10 mm) were obtained from each embedded block and placed onto glass slides. Finished tissue slides were stained utilizing a modified Heidenhain's Aniline-Blue method (Coolidge & Howard, 1979) to examine the maturation stages of gametocytes and embryos. Slides were examined under an Olympus BX40 compound microscope coupled to an Olympus DP26 digital microscope camera. Images were captured utilizing Olympus cellSens 1.7 imaging software. The sexual pattern, gametogenic cycle and fecundity of each species were determined by observing the gametocyte development throughout the collection year. Gamete stages were characterized according to Szmant-Froelich, Reutter & Riggs (1985). Oocyte sizes were obtained using cellSens, by taking perpendicular measurements at the cell's widest points. Cell length and width measurements were used to calculate geometric area. Fecundity was assessed by counting oocytes per mesentery (I. sinuosa n = 120; I. rigida n = 60) and per polyp (I. sinuosa n = 10; I. rigida n = 5) on histologic cross-sections during months with the highest proportion of mature oocytes (I. sinuosa April 2001 n = 5; I. rigida May 2001 n = 5).
In April 2012, several presumed gravid colonies of each species were collected and placed in an open seawater aquarium system to observe planulation. Two colonies of each species were placed within six-gallon aerated aquariums under continuously circulating seawater and daylight synchronized lights. Specimens were placed under mesh-lined PVC pipes allowing water to freely circulate. Traps were checked daily for larvae over a 90-day period.

Statistical analyses
Results are expressed as means ± standard error. All statistical tests were performed using the RStudio 0.99.484 software platform (R Studio Team, 2015) using the stats package (R Development Core Team, 2015). Normality was assessed using the Shapiro-Wilk test performed with the R function shapiro.test. Equality of variance was tested using the F test performed with the R function var.test. Differences in fecundity were tested by means of a Wilcoxon rank sum test with continuity correction performed with the R function wilcoxon.test.

I. sinuosa
Stage I oocytes are small (78.92 ± 13.15 mm 2 ), stain pink and are characterized by sparse cytoplasm and prominent nuclei (Fig. 4A). Oocytes originate within the linings of the mesoglea in the central regions of the mesenteries. Stage II oocytes are larger than stage I cells (144.54 ± 43.19 mm 2 ), exhibit prominent nuclei and abundant cytoplasm (Fig. 4A). Stage III oocytes are larger than stage II (264.51 ± 37.24 mm 2 ), tend to have a round shape, stain pink or red, and are characterized by many cytoplasmic globules which produce a grainy appearance (Fig. 4B). Stage IV oocytes are larger and boxier than stage III (376.69 ± 73.20 mm 2 ). This stage is characterized by dark staining nuclei and large globules in the cytoplasm (Figs. 4C-4E).
No stage I spermaries were found, suggesting this stage occurs briefly and/or is difficult to differentiate using the current method. Stage II spermaries form small poorly defined bundles which form in the mesenteries surrounding oocytes (Fig. 4C). Stage III spermaries form small sacs with well-defined borders (Fig. 4D) and contain bright red staining spermatids. Stage IV spermaries stain dark red and are larger than stage III. Tails visible on spermatozoa at high magnification are indicative of stage V spermaries (Fig. 4E). Spermary sizes were not measured.
Stage I planulae are approximately the same size as stage IVoocytes (404.07 mm 2 ) and stain pink. During this stage, zooxanthellae become visible within the planulae. Stage II planulae (455.45 ± 32.84 mm 2 ) are characterized by an outer layer composed of columnar cells which contain nematocysts and cilia (Fig. 4F). Developing mesenteries can be seen within the gastrodermis of stage III planula (501.98 ± 44.68 mm 2 ). Stage IV planula were not observed.
The gametogenic cycle of I. sinuosa is summarized in Fig. 5. Weekly sea surface temperature measurements taken during the collecting period are included for reference ( Fig. 5A). Oogenesis in I. sinuosa lasts approximately 11 months (Fig. 5B) Stage I-III planulae were observed in histologic sections during April 2001 (Fig. 5D). The identification of planulae on tissue sections coincided with a sharp decrease in the proportion of colonies containing mature (IV) oocytes. No larvae were collected from specimens placed in aquaria for observation.

I. rigida
Stage I oocytes are very small (72.97 ± 15.75 mm 2 ) and are characterized by sparse cytoplasm and a large nucleus. Stage II oocytes are larger than stage I cells (101.25 ± 23.09 mm 2 ), are ovoid shaped and feature a prominent nucleus and nucleolus (Fig. 6A). A pink-staining nucleus and red nucleolus can clearly be identified in many stage III oocytes (148.77 ± 49.35 mm 2 ) (Fig. 6B). Stage IV oocytes are large (190.40 ± 45.18 mm 2 ), irregularly shaped and contain large vacuoles in the ooplasma which give it a grainy appearance (Figs. 6C and 6D).
Stage I spermaries were not detected in I. rigida. Stage II spermaries were observed forming adjacent to stage III eggs (Fig. 6B). Spermaries typically adopt a spherical shape and often form in series resembling a string of beads (Figs. 6B and 6C). Stage III spermaries form small oblong sacs and stain red (Fig. 6C). Stage IV spermaries are densely packed with sperm, have irregular shapes, stain dark red to brown. Stage V spermaries stain darker than stage IV (Fig. 6E) but are characterized by tails on spermatozoa under high magnification. No measurements were collected for spermaries.
Stage I planulae are approximately the same size as stage IV oocytes (approximately 324.01 ± 71.64 mm 2 ), stain pink, and contain zooxanthellae in the epidermis. Zooxanthellae were observed within planula beginning at this stage. Stage II planulae are   Fig. 7. Weekly sea surface temperature measurements taken during the collecting period are included for reference (Fig. 7A). Oogenesis in I. rigida lasts approximately 10 months (Fig. 7B)

Oocyte size
Measurements of oocyte geometric area in I. sinuosa (range 43.94-463.79 mm 2 ) show an increase in the size of oocytes as maturity progresses from April through March (Fig. 9A). Mean geometric area is lowest during the month of June 2000 (97.22 ± 28.85 mm 2 ) and greatest during February 2001 (333.95 ± 74.32 mm 2 ). The appearance of planulae in histological sections during the month of April 2001 (459.07 ± 45.83 mm 2 ) (range: 404.07-548.49 mm 2 ) coincides with a sharp decrease in mean geometric area of oocytes compared to the previous month (285.68 ± 96.46 vs. 143.28 ± 84.07 mm 2 ). Measurements of oocyte geometric area in I. rigida (range 43.31-307.35 mm 2 ) also show a trend of increasing oocyte size as maturity progresses from August through June (Fig. 9B). Mean geometric area is lowest during the month of September 2000 (68.35 ± 17.04 mm 2 ) and greatest during June 2000 (210.54 ± 42.90 mm 2 ). Mean planulae area was greatest during the month of July 2000 (909.48 ± 250.56 mm 2 ) and ranged from 241.66-1,183.96 mm 2 . Mean oocyte geometric area was greater in I. sinuosa than in I. rigida (Wilcoxon-rank sum test, W = 43,911, p < 2.13 Â 10 -13 ); however, mean planulae geometric area

DISCUSSION
Microscopic observations indicate that both I. sinuosa and I. rigida are simultaneous hermaphrodites (gametes of both sexes are present in a single individual at the same time). Gametes of both sexes are produced adjacent within the same mesentery (dygonism) in both species. Both species are brooders (bear live young) which transfer endosymbiotic zooxanthellae directly from parent to offspring. Both species are characterized by a single annual gametogenic cycle. This study represents the first description of the sexual characteristics of I. rigida and contradicts observations by Duerden (1902) which label I. sinuosa as a gonochoric species. The incorrect classification of I. sinuosa as the sole gonochoric outlier within the traditional Mussidae was a contrasting element in a group which is otherwise uniformly hermaphroditic  Duerden (1902), Fadlallah (1983, Szmant (1986), Richmond & Hunter (1990) Szmant (1986), Richmond & Hunter (1990), Soong, 1991, Steiner (1995 (Duerden, 1902;Fadlallah, 1983;Richmond & Hunter, 1990). This study confirms the dominant pattern of sexual reproduction described for Mussid corals (Baird, Guest & Willis, 2009) and provides further support for conserved reproductive patterns within coral families (Harrison, 2011). Traditional morphology-based classifications are being restructured by designating systematic affinities using molecular methods in combination with morphometric analyses. The traditional Mussidae family has recently undergone extensive restructuring by separating Indo-Pacific Mussids from their Atlantic counterparts which are more closely related to some members of the family Faviidae (Fukami et al., 2004;Fukami et al., 2008;Budd et al., 2012). The resulting 'modern' Mussidae (clade XXI) is composed of the genera Mussa, Isophyllia, Mycetophyllia, and Scolymia (Atlantic) under the Mussinae subfamily and Favia (Atlantic), Colpophyllia, Diploria, Pseudodiploria, Manicina and Mussismilia under the Faviinae subfamily. Under the new classification, hermaphroditism has been exclusively documented within all genera of the subfamily Mussinae: Mycetophillia (Szmant-Froelich, 1985;Morales, 2006), Scolymia (Pires, Castro & Ratto, 2002;E. Weil, 2016, unpublished data) and Mussa (Steiner, 1993) and within the subfamily Faviinae: Favia (Soong, 1991) (Johnson, 1992), Mussismilia (Pires, Castro & Ratto, 1999) (Table 1). Mode of development within the modern Mussidae is mixed; both brooding and spawning species are present. Brooding has been documented within Mycetophyllia (Morales, 2006), Scolymia (Pires, Castro & Ratto (2002); E. Weil, 2016, unpublished data), and Manicina (Johnson, 1992). Broadcast spawning occurs in Colpophyllia (E. Weil, 2016, unpublished data), Diploria (Weil & Vargas, 2010), Pseudodiploria (Weil & Vargas, 2010, and Favia (Soong, 1991). Sexual mode exhibits more plasticity than sexuality (Van Moorsel, 1983;Harrison, 1985): contrasting modes of development exist within families and even within genera (Harrison, 2011). Szmant (1986) suggested that sexual mode is potentially a function of habitat stability, where successful recruiters would be small, rapidly maturing species, which produce many offspring over short periods but subject to high mortality rates. Thus, the sexual modality of species occupying unstable habitats would gravitate towards brooding because it increases the chances of successful recruitment by reducing gamete and larval mortality even in low population densities. Edinger & Risk (1995) on noting a correlation between brooding and eurytopy, hypothesized that brooding corals may preferentially survive in unstable habitats due to higher recruitment success. The benefits provided by the brooding modality may partially explain why, in recent decades, brooding corals have begun to dominate some Caribbean reefs following degradation by natural and anthropogenic disturbances (Hughes, 1994;Mumby, 1999;Knowlton, 2001;Irizarry-Soto & Weil, 2009).
The close proximity of oocytes and spermaries within the same mesentery (dygonism) in I. sinuosa and I. rigida suggests that it is possible that self-fertilization can occur in these species. Generally, self-fertilization is not a favored method of fertilization in corals due to possibility of inbreeding depression (Knowlton & Jackson, 1993). Selfing, however, is thought to be advantageous in sessile hermaphrodites which are ecologically distant from other mates and may have limited access to gametes of the other sex, providing a viable alternative for successful fertilization (Ayre & Miller, 2004;Darling et al., 2012;Sawada, Morita & Iwano, 2014). These corals may then switch to sexually produced larvae as population sizes increase (Ayre & Resing, 1986). Selfing has been documented in the brooding corals Seriatopora hystrix (Sherman, 2008), Favia fragum and Porites astreoides (Brazeau, Gleason & Morgan, 1998).
The duration of the gametogenic cycle is similar in I. sinuosa and I. rigida (11 and 10 months, respectively). Long oocyte generation times, differential gamete maturation, and long brood retention times in Isophyllia suggest the possibility of multiple brooding events during a single gametogenetic cycle. This strategy may increase reproductive output due to space limitations within polyps. A single annual gametogenetic cycle is the dominant pattern in most broadcasting corals such as Orbicella, Montastraea, Diploria, Porites, Acropora, Siderastrea (Szmant, 1986;(Vargas-Ángel & Thomas, 2002;Weil & Vargas, 2010) and brooding Caribbean corals like Porites and Mycetophyllia (Szmant, 1986;Soong, 1993;Vermeij et al., 2004;Morales, 2006). Multiple spawning events have been documented in Acanthastrea lordhowensis (Wilson & Harrison, 1997) and cannot be ruled out in these species.
Both species differ in the timing of oogenesis and planulogenesis events by various months which suggests that opportunities for hybridization between both species are limited. The dates of onset of oogenesis in both species (May in I. sinuosa and August in I. rigida) coincide with warm local sea surface temperatures suggesting seasonal synchronization of the gametogenic cycle. In I. sinuosa, planulae were observed in histologic sections during April 2001 which suggests that fertilization occurred during early April (most recent Full Moon: April 9). In I. rigida, planulae were observed in June 2000 which suggests a fertilization date in late May (most recent Full Moon: May 6, 2001). Various environmental factors have been shown to correlate with coral reproductive cycles and may play a role in their synchronization, including sea temperature, salinity, day length, light/dark cycles and tidal cycles (Harrison & Wallace, 1990). Van Woesik, Lacharmoise & Köksal (2006) showed experimentally that some coral spawning schedules correlate strongly with solar insolation levels prior to gamete release, however, water temperatures are highly influential in determining actual gamete maturity. van Woesik (2009) also demonstrated a positive correlation between the duration of regional wind calm periods and the coupling of mass coral spawnings. Studies with the brooding coral Pocillopora damicornis revealed that synchronization of larval production was lost under constant artificial new moon and full moon conditions, demonstrating that planulation in some species is linked to nighttime irradiance (Jokiel, Ito & Liu, 1985).
Acquisition of the endosymbiont Symbiodinium in Isophyllia occurs directly from parent to offspring (vertical transmission), a characteristic strongly linked to the brooding modality (Baird, Guest & Willis, 2009). Vertical symbiont transmission may be advantageous by providing larvae with various Symbiodinium genotypes which may improve their ability to recruit successfully and grow in different environmental conditions (Padilla-Gamiño et al., 2012). Brooded larvae are capable of motility immediately or shortly after planulation (Fadlallah, 1983), in contrast to broadcast spawned propagules which are positively buoyant and may take between 12-72 h to become motile (Baird, Guest & Willis, 2009). By avoiding the surface, brooded larvae may better avoid exposure to high levels of solar radiation which may overwhelm the photosynthetic capacities of zooxanthellae producing oxygen radicals (Tchernov et al., 2004) and cause tissue damage and mortality (Lesser et al., 1990). However, under high temperature conditions, larvae of corals with vertical symbiont transmission may suffer higher oxidative stress and tissue damage, suggesting that these corals may be more vulnerable to the effects of ocean warming (Yakovleva et al., 2009).
There is increasing evidence that sexual reproduction in corals is highly susceptible to natural and anthropogenic stressors that reduce fecundity, fertilization success, and larval survival (Harrison & Wallace, 1990;Harrison, 2011). Increases in sea surface temperatures as a consequence of global warming have produced widespread coral bleaching events and disease outbreaks with massive mortality of susceptible individuals. This worldwide decline of coral reefs underscores the need for understanding sexual reproduction in corals as the only mechanism capable of safeguarding their future. Sexual recombination is an important prerequisite for the selection of individuals which are to be able to adapt to the pressures of a changing environment. A greater understanding of the mechanisms and variables in sexual reproduction in corals, in combination with knowledge of the taxonomy and variability of the species, is essential for any coral reef management strategy (Harrison & Wallace, 1990).


s2fieldsofstudy: Biology Medicine

text:
Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes

Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.

There is recent strong evidence to suggest that immune cells such as monocytes and macrophages play important roles in the overall disease pathology. While it is likely that repeated cycles of lung epithelial injury followed by accumulation and activation of fibroblasts in IPF are significant drivers of disease pathogenesis, recent studies suggest that activated macrophages are also likely key contributors 8 . For example, injured type II alveolar epithelial cells produce cytokines leading to accumulation of activated macrophages at fibroblastic foci 9 . This activated macrophage population also produces transforming growth factor beta (TGF-β ) and CCL18, potent pro-fibrotic cytokines that are key mediators of lung fibrosis. CCL18 is produced preferentially by alternatively activated macrophages and high serum levels of this cytokine in IPF patients is associated with a higher incidence of disease progression 10 . Serum CCL18 has also been shown to predict lung disease worsening in systemic sclerosis 11 . Finally, a recent study found that surfactant protein D (SP-D) deficiency leads to macrophage infiltration and the production of the pro-fibrotic cytokines, TGF-β and platelet derived growth factor AA (PDGF-AA) in the BLM lung fibrosis model 12 .
Cysteine cathepsins are a group of proteases with elastinolytic and collagenolytic activities that are involved in various aspects of ECM remodeling 13 . Of the 11 members of this cysteine protease family, cathepsins B, L and S contribute to the degradation of several types of collagen and elastin in the extracellular space 14 . These proteases are also secreted into the extracellular space and the bronchoalveolar lavage (BAL) fluid in pulmonary disorders 15 . Furthermore, the activities of these proteases in lung tissue homogenates and activated alveolar macrophages are elevated during tissue regeneration and remodeling 16 . Because cathepsins are highly expressed in activated macrophages 17,18 , they are potential diagnostic and therapeutic targets in IPF disease pathogenesis.
Reagents that can detect activated forms of the cysteine cathepsins are valuable tools for highlighting sites of disease pathology involving macrophage infiltration [19][20][21] . We have developed small molecule optical imaging probes that specifically report on the activity of cysteine cathepsins 22 . These probes can be used to image populations of activated macrophages that have high expression of the cysteine cathepsins. We have also demonstrated that positron emission tomography (PET) versions of cathepsin probes can be used for non-invasive imaging of cancer margins as the result of the high content of activated macrophages within solid tumours 23 .
In this study, we utilize the bleomycin-induced murine model of lung fibrosis, tissues from human clinical samples and first-in-human imaging studies to demonstrate the value of non-invasive imaging of cysteine cathepsin activity to understand the role of these proteases in IPF disease pathobiology. Our results confirmed an increased uptake of the imaging agent in fibrotic lesions suggesting a role for activated macrophages in the pathobiology of the human disease.

Results
Generation of molecular probes for dual optical/PET imaging of activated macrophages. We have developed several classes of activity based probes (ABPs) targeting cysteine cathepsins [18][19][20]22,24 . In particular, the probe GB123, containing a 2,6-dimethylbenzoic acid-derived acyloxymethyl ketone (AOMK) electrophile has been validated in numerous in vivo optical imaging applications and was shown to target cathepsins B, S and L 19,[25][26][27] . Recently, we demonstrated that a cathepsin ABP containing a phenoxymethyl ketone (PMK) electrophile, BMV109, has enhanced in vivo properties and broad reactivity towards cathepsins X, B, S and L 22 and can be used for imaging of tumour margins in vivo 21 . Based on these highly successful probes, we designed a dual labelled ABP, BMV101, that could be used for both optical and PET/CT imaging applications (Fig. 1). This new probe contains a Cy5 near infrared (NIR) fluorophore for optical detection and a NOTA chelator for labelling with 64 Cu, Al 18 F or 68 Ga for PET/CT imaging applications. Importantly, the new probe, BMV101, performed nearly identically to BMV109 and GB123, with respect to labelling of cathepsins B, S and L in intact cells ( Supplementary Fig. S1).
Imaging disease progression in the bleomycin-induced model of lung fibrosis. We first performed a study with the optimized optical probe, BMV109, to determine if cysteine cathepsins could be used as markers of pulmonary fibrosis in the lung. Bleomycin has been used extensively to model pulmonary fibrosis in rodents, with direct administration into the lungs being highly effective in eliciting fibrosis 28 . We assessed the extent of probe accumulation in the lungs, focusing on a time course between 7 and 14 days ( Fig. 2A), a time period in which immune cell infiltration and initiation of fibrosis takes place 29 . Disease burden was evident in the bleomycin treated group at both time-points after bleomycin administration as measured by total body weight loss and increased lung weight ( Supplementary Fig. S2A). Lung tissue at day 7 and at day 14 showed histological evidence of pulmonary fibrosis, at both time points and was greatest at day 14 ( Supplementary Fig. S2B). We were also able to confirm the onset of fibrosis by increased lung collagen content at Day 14 ( Supplementary Fig. S2B).
For optical imaging studies, we injected mice with the BMV109 probe via tail vein at each time-point and non-invasively quantified Cy5 fluorescence in the lung region 4 hours after probe injection ( Fig. 2A). At Day 7, bleomycin-treated mice showed significantly higher fluorescence signal in the lungs when compared to the saline controls. Signal intensity further increased at Day 14. Probe signal was also detected in the liver and kidney of both groups due to clearance of the probe. We found that levels of fluorescence in the excised lungs directly correlated with the levels observed using non-invasive optical imaging methods (Fig. 2B). Subsequent analysis of lung tissues by SDS-PAGE confirmed that the primary targets of the probe in these tissues were cathepsins X, B, S and L, and that levels of labelling of these targets correlated with the extent of disease burden and intensity of signals observed by non-invasive imaging (Fig. 2C).
Immune cell contribution to disease progression in the bleomycin lung fibrosis model. Brief exposure to bleomycin causes epithelial cell apoptosis, activating an inflammatory wound-healing response, ultimately leading to pulmonary fibrosis. We sought to further characterize the immune cell infiltrate and define the distinct cell populations labelled by BMV109 during bleomycin-induced fibrosis. After bleomycin exposure, probe labelling in the lung co-localized specifically with the macrophage marker CD68, with the intensity of the probe signal increasing from Day 7 to Day 14 as disease burden increased ( Supplementary Fig. S2C). Flow cytometric analysis allowed us to further characterize the population of infiltrating leukocytes in diseased lung, with particular focus on the probe labelled macrophage population. Bronchoalveolar lavage (BAL) fluid from mice post bleomycin exposure had significantly more leukocytes compared to saline controls. Macrophage populations dominated the immune infiltrate across all groups tested. Neutrophils were enriched in control and bleomycin treated mice on day 7, but declined by day 14. Day 14 was marked by a significant increase in other immune cells, consistent with lymphocytes (by forward and side scatter profiles), corresponding to increased inflammation ( Supplementary Fig. S2D). BMV109 staining was highly enriched in the F4/80+ /CD11c+ macrophage populations, with the intensity of probe per cell (mean fluorescence intensity, MFI) greater in bleomycin-treated mice at both time points, indicating increased cathepsin activity in these cells (Fig. 3A). Intracellular flow cytometric analysis revealed an increased macrophage expression of pro-inflammatory IL-12 at day 7, in BAL fluid that was not sustained at day 14 ( Fig. 3B). Furthermore, a shift to the pro-fibrotic response was evident, with increased M2-like macrophage associated IL-10 production and CD206 expression.
Coordinate or discordant regulation of several cytokines and chemokines are reported to correlate with IPF disease progression 30 . To determine if these markers mimicked levels observed in the human condition, we performed multiplexed cytokine profiling of plasma from bleomycin-treated mice at Day 7 and Day 14 (Fig. 3C). We found that the cytokines IL-1β , IL-10 and IL-2 increased from Day 7 to Day 14. Similarly, a downstream target of IL-1β , IL-17 increased from the early to late stages of the disease. This cytokine plays a role in mediating the fibrotic response 31 . Markers of the Th2, pro-fibrotic immune response, IL-4 and IL-5, were low at Day 7 but increased with disease progression at Day 14. Overall, the changes in the levels of cytokines and chemokines upon induction of fibrosis in the mouse model were consistent with those observed in human IPF samples 32,33 . PET/CT and optical imaging of cathepsin activity in the bleomycin lung fibrosis model. We next applied 64 Cu-labeled BMV101 to the bleomycin model. We chose this radionuclide because it has a relatively long half-life (12.7 hrs) and therefore could be used for imaging at extended time points (i.e. 24 hr) after probe injection. We performed optical/PET-CT imaging studies with 64 Cu-BMV101 at the same time points post bleomycin treatment that we used for the optical probe except we added a Day 21 time point to cover later stage progression of the disease ( Fig. 4 and Supplementary Fig. S2,3). Again, disease burden was evident in the bleomycin treated groups as measured by total body weight loss and increased lung weight (Supplementary Fig. S3A). We performed small animal PET-CT imaging and observed increased probe signal at Day 7 and Day 14 in lungs of bleomycin treated mice relative to control mice (Fig. 4A). Furthermore, this level increased from Day 7 to Day 14 as macrophage infiltration increased. At Day 21, we observed reduced probe signal in the bleomycin treated lungs, likely due to the fact that in the rodent fibrosis model, immune cell infiltration decreases over time 34 . Biodistribution studies confirmed that the probe accumulated in the blood, liver, heart and kidney, but showed the most significant increased accumulation in the lungs of the bleomycin treated mice compared to controls ( Supplementary Fig. S5A). Specific probe uptake into lungs was confirmed by quantification and comparison of lung to liver and lung to muscle ratios at each time-point ( Supplementary Fig. S5B). Since the probe has dual optical and PET labels, we were also able to visualize probe signals in the lungs by non-invasive optical imaging and by ex vivo imaging of the lungs (Fig. S4). Day 7 and Day 14 mice with lung fibrosis showed significantly higher optical probe signal in the lung when compared to the controls by both non-invasive and ex vivo imaging ( Supplementary Fig. S4), consistent with the PET-CT results. Subsequent analysis of lung tissues by SDS-PAGE revealed increased levels of active cathepsins, dominated by cathepsin B and S, in the bleomycin-induced lung samples relative to the controls (Fig. 4B). Non-invasive and ex vivo analysis of Day 21 samples showed decreased lung cathepsin expression, which correlated with a decrease in macrophage infiltration ( Supplementary Fig. S4).  This was further confirmed by histology and immunofluorescence analysis ( Supplementary Fig. S3B and S3C). Taken together, these results confirm that the non-invasive PET imaging signals correlated with the onset of fibrosis at days 7 and 14 and correlated with the presence of activated macrophages in the lungs.

Topical labelling of human lung tissues from patients with idiopathic pulmonary fibrosis.
We have recently shown that small molecule ABPs can be used to topically label active cathepsins in excised tissues 21,35 . We therefore topically labelled sectioned frozen tissue samples from IPF patients undergoing surgical lung biopsy or lung transplantation with the optical probe BMV109 (Fig. 5, S6). We also co-stained the tissues with the macrophage marker (CD68). These data confirmed labelling of macrophages that are actively expressing cysteine cathepsins and that the signals for active cathepsins were specifically found at sites of fibrotic lesions. Thus, cathepsins may be ideal biomarkers for monitoring of disease activity in IPF.

Application of an activity based PET imaging probe in patients with idiopathic pulmonary
fibrosis. We chose to use 68 Ga as our radionuclide for first-in-human trials since it has been used extensively in clinical imaging and is relatively easy to generate. However, it has a short half-life (68 min) and therefore required imaging within the first few hours of probe administration. Prior to translation into studies in humans, we tested the effectiveness of 68 Ga-BMV101 in the bleomycin lung fibrosis model ( Supplementary Fig. S7). These results confirmed that it produced similar results to 64 Cu-BMV101, with increased probe signal observed in lungs of bleomycin treated mice.
For the human clinical studies, we recruited six subjects and three controls with healthy lung function (Supplementary Table S1). Of the six patients with lung fibrosis, three were confirmed as having idiopathic pulmonary fibrosis by multidisciplinary review according to ATS/ERS/JRS/ALAT criteria 3 while the remaining three were unclassifiable pulmonary fibrosis. Subjects were between the age of 51 and 76, were predominantly former smokers, and had varying levels of physiological impairment. The 68 Ga-BMV101 PET-CT probe was injected intravenously and images were collected at 2.5 hours post probe administration. These data demonstrated that the probe showed increased accumulation in regions of fibrosis in the lungs of the three patients with IPF, with no significant increase in probe accumulation in the unclassified fibrosis patients relative to the normal lung (Fig. 6). Importantly, there was also no significant increase in probe accumulation in the liver of the patients with IPF or lung fibrosis compared to the healthy controls. Overall, the ratio of probe signals in lung compared to muscle were high (4.36 for IPF patients and 1.37 for normal controls; Table S) with more than 3 times greater accumulation of the probe in lungs of patients with IPF. Taken together, these results confirm that the probe is a viable PET contrast agent that accumulates in regions of lung fibrosis in patients with IPF. The data also suggest that the bleomycin-induced fibrosis in the mouse correlates with the pathobiology of human patients with IPF, and suggest that macrophages may be important regulators of disease progression in humans.

Discussion
Concerted efforts have recently been made to develop new technologies for early disease monitoring to facilitate diagnosis and therapeutic intervention for IPF. Optical and PET imaging has great promise and potential application in this respect 22 . Activity based probes (ABPs) generate a signal in response to covalent modification of a specific enzymatic target and are therefore useful as contrast agents for imaging. Cysteine cathepsins expressed in antigen presenting cells such as macrophages, are promising biomarkers of macrophage activation, with cathepsins B and S in particular, implicated in lung disease 36,37 . Recent evidence suggests that these immune cells play important roles in the overall disease pathology of IPF 8 . For these reasons, we hypothesized that ABPs that target cysteine cathepsins would specifically highlight infiltrating macrophages that contribute to the pathobiology of IPF, and that these ABPs could have broad value for non-invasive monitoring of disease activity. We further expect that these probes might also allow for early diagnosis with increased sensitivity compared to HRCT 38 . Currently, there have been no reported non-invasive imaging clinical studies of any contrast agent in patients with IPF.
Here we describe the application of cysteine cathepsin activity-based probes to the rodent model of lung fibrosis and in human IPF lungs. The relevance of the bleomycin mouse model to IPF has been questioned because it is thought to mimic acute lung injury followed by scar formation, rather than abnormal wound healing or progressive fibroproliferation. Also, with the direct administration of bleomycin to the lungs, the initial phase of bleomycin-induced injury is primarily pro-inflammatory with an influx of neutrophils and mononuclear infiltrate 30 , which has not been observed in IPF. Although the bleomycin model has been criticized for its speed of disease onset and durability of fibrosis, it remains a robust, well-established, reproducible model of lung fibrosis that can reveal potential mechanisms underlying the human disease 30,31 . Our results presented here further confirm that this model produces many of the same hallmarks of human IPF disease pathology, especially with respect to the activation and accumulation of macrophages and the production of hallmark cytokines. Specifically, our data shows that IL-1β , IL-10, IL-2, were key cytokines that increased from Day 7 to Day 14 in the murine model, consistent with what has been observed in primary patient samples 9 . Thus, for the purposes of validating imaging probes that aim to report on relevant aspects of the immune program in IPF disease pathogenesis, this model remains highly valuable.
The first-in-human results for the 68 Ga-labeled probe provide strong support for the notion that activated macrophages are regulators of the pathobiology of IPF disease progression. Our clinical results mirror our findings in the bleomycin mouse model of fibrosis where we see accumulation of macrophages with high cathepsin activity both at early onset and once the fibrotic process has been fully established. This again suggests that the bleomycin model proves valuable information that is relevant to the human disease, with respect to immune cell involvement. Furthermore, the fact that we do not see significant probe accumulation in patients with unclassified lung fibrosis provides some support for the potential to use cathepsin probes to stratify patients with IPF such that they can be followed and eventually treated with new therapies that are currently in clinical trials. We believe that additional follow up imaging trials on a larger number of patients, coupled with collection of BAL fluids for analysis of levels of activated macrophages in the lungs, will allow a more clear understanding of the importance of activated macrophage populations with respect to IPF disease severity, progression and response to treatment. While our initial results with the dual optical/PET probe are promising both in the preclinical animal models and in human patients with IPF, we noted that the overall signal to noise ratios were rather poor (see Tables S2-4). Specifically, when comparing lung to liver signals we obtained values below a value of 1. This is likely do to high baseline cathepsin activity in the liver and also relatively high blood pooling of the probe. This explanation was further supported by the fact that lung to muscle ratios were substantially higher. We also observed high PET signals in the blood (Fig. S5). This blood pooling effect is likely due to the highly hydrophobic/aromatic nature of the probe. Therefore, we are currently working to identify a more effective probe backbone structure that is still recognized by the protease targets but that is more highly hydrophilic and charged. This should both help to reduce the blood retention of the probe and also increase solubility and formulation properties which should facilitate further clinical studies.
In conclusion, we report the development of a new bimodal optical/PET probe and its use as an imaging agent for monitoring the pathobiology of IPF. Having a dual label on the probe allows for initial PET imaging and subsequent optical analysis by in vivo and in vitro methods. Importantly, the use of a PET/optical probe, for the first time, provided a means to non-invasively image regions of lung fibrosis and correlate findings in the mouse model of fibrosis with human IPF disease. We find that the early stages of fibrosis in the mouse model involve macrophage responses that peak early in the onset of fibrosis (i.e. day 15). In humans, we find that the probe accumulates in regions of fibrosis that are more likely late events that may involve persistent, unresolved injury around established fibrotic lesions. Thus, our probe could be highly valuable for assessing overall disease activity, even in patients who have advanced disease. Furthermore, these results suggest that our probe has the potential to be applied in the clinic to facilitate study of the cellular and molecular events that drive IPF pathology. They are also likely to be useful preclinical and clinical tools for other diseases that involve contributions from activated immune cells that express the cysteine cathepsins.

Materials and Methods
Synthesis of Probes. Details of the probe synthesis, radiolabeling and compound characterization can be found in the supplemental methods section.
Animals and Model Induction. Female C57BL/6J mice weighing 20-25 g were purchased from Jackson Laboratories (Bar Harbor, ME). To induce pulmonary fibrosis, mice were treated on day 0 with 1.5 unit/kg body weight of bleomycin (MP Biomedicals LLC) by endotracheal instillation as previously described 39 ; control animals received sterile saline. At the indicated times (day 7 and day 21), the probe BMV109 was injected at a dose of 10 nmol in 10% DMSO/PBS, 4 hours prior to harvest, and mice were imaged non-invasively using the IVIS 100 system. Animals were sacrificed under isoflurane anesthesia and BAL fluid extracted as previously describe 40 . Lungs were then removed, imaged ex vivo using an FMT system. Samples of lung tissue and cells were collected for further analysis, as described below. A total of 10 animals were used for each time point, divided equally between control and bleomycin-treated groups and not randomized. This sample size was chosen based on knowledge that some animals may not survive the course of the experiment and that a cohort of at least 3 animals per group are required in order to get statistical significance in the results. Investigators conducting the trial were not blinded as to what groups were being studied unless otherwise stated. All mouse experiments were approved by the Stanford Administration Panel on Laboratory Animal Care and strictly followed their specific guidelines.
Murine PET In Vivo Imaging. Mice were intravenous injected with 100 μ Ci of 64 Cu-BMV101 and imaged after 1 h, 2 h, and 4 h by using Inveon small-animal PET/CT (Siemens). Briefly, a CT anatomic image scan was acquired (80 kV, 500 μ A) with a pixel size of approximately 0.1 mm. After CT imaging, whole-body PET imaging was performed with 5 min static scan. The PET images were reconstructed using the ordered-subsets expectation maximization 3-dimensional algorithm based on CT attenuation and analyzed using the Inveon Research Workplace (IRW) software (Siemens). PET voxel size was 0.796 × 0.861 × 0.861 mm, for a total of 128 × 128 × 159 voxels. After the 4 h PET/CT imaging, the mouse was injected with 100 μ L BMV101 (10 mM) and imaged for fluorescence signal by IVIS200 (Perkin Elmer) at 1 h later. Then PET/CT quantify assay was performed, and tissue radioactivity was calculated and expressed as decay-corrected percentage injected dose per gram of tissue (%ID/g). Investigators conducting the trial were blinded as to what groups were being imaged.
Tissue and cell labeling and analysis. Details of the tissue and cell preparation, probe labeling and analysis by microscopy and flow cytometry can be found in the supplementary methods section.

Patient Recruitment. This study was approved by the Institute Review Boards of Peking Union Medical
College Hospital and conducted from December 2014 to April 2015 and was carried out in accordance with the approved guidelines. Nine patients were enrolled in this study after obtaining written informed consent. The subjects were deemed to have IPF or IPF-related condition based on their clinical history, physical examination, standard blood and urine tests, CT and electrocardiogram. Subjects were contacted by telephone approximately 24 h after 68 Ga-BMV101 administration, for adverse events monitoring. Subjects included were between the ages of 35 and 80 and had fibrotic interstitial lung disease as defined by the presence of reticulation, traction bronchiectasis and honeycombing on high-resolution computed tomography (HRCT) imaging of the chest. Controls without lung disease were recruited from patients undergoing PET-CT scans for non-pulmonary indications. Exclusion criteria included pregnancy, lactation, the presence of co-existing emphysema on HRCT, and impaired renal or liver function. Human subjects research was approved by the ethics review committees of Stanford University Medical Center (Stanford, CA, United States) and Peking Union Medical College (Beijing, China), and all participants provided informed consent.
Scientific RepoRts | 6:19755 | DOI: 10.1038/srep19755 Statistical Analysis of Data. Statistics were performed using the data analysis package within GraphPad Prism 6.0 for Windows (GraphPad Software, San Diego, CA, USA). For the human studies, SUVmax values from subjects and controls were compared using the Wilcoxon Mann Whitney U test to account for non-normally distributed data. Statistical significance was defined as a p value < 0.05. Unless otherwise stated, tests comparing two means are a Student's t-test, with equal variance assumed. Error bars indicate standard error of the mean (SEM) unless otherwise stated.