Although immunotherapies that target CD20 on most non-Hodgkin lymphoma (NHL) cells have improved patient outcomes, current therapies are inadequate because many cases are, or become, refractory or undergo relapse. Here, we labelled the third-generation human anti-CD20 antibody ofatumumab with 177Lu, determined the in vitro characteristics of [177Lu]Lu-ofatumumab, estimated human dosimetry, and assayed tumor targeting and therapeutic efficacy in a murine model of disseminated NHL. Methods: CHX-A″-diethylenetriaminepentaacetic acid-[177Lu]Lu-ofatumumab was prepared. We evaluated radiochemical yield, purity, in vitro immunoreactivity, stability, (n = 7), affinity, and killing of CD20-expressing Raji cells (n = 3). Human dosimetry was estimated from biodistribution studies as percentage injected activity per gram using C57BL/6N mice. Tissue and organ biodistribution was determined in R2G2 immunodeficient mice with subcutaneous Raji-cell tumors. Therapy studies used R2G2 mice with disseminated human Raji-luc tumor cells (n = 10 mice/group). Four days after cell injection, the mice were left untreated or were treated with ofatumumab, 8.51 MBq of [177Lu]Lu-IgG, or 0.74 or 8.51 MBq of [177Lu]Lu-ofatumumab. Survival, weight, and bioluminescence were tracked. Results: Radiochemical yield was 93% ± 2%, radiochemical purity was 99% ± 1%, and specific activity was 401 ± 17 MBq/mg. Immunoreactivity was substantially preserved, and more than 75% of 177Lu remained chelated after 7 d in serum. [177Lu]Lu-ofatumumab specifically killed Raji-luc cells in vitro (P < 0.05). Dosimetry estimated that an effective dose for human administration is 0.36 mSv/MBq and that marrow may be the dose-limiting organ. Biodistribution in subcutaneous tumors 1, 3, and 7 d after [177Lu]Lu-ofatumumab injection was 11, 15, and 14 percentage injected activity per gram, respectively. In the therapy study, median survival of untreated mice was 19 d, not statistically different from mice treated with 8.51 MBq of [177Lu]Lu-IgG (25 d). Unlabeled ofatumumab increased survival to 46 d, similar to 0.74 MBq of [177Lu]Lu-ofatumumab (59 d), with both being superior to no treatment (P < 0.0003). Weight loss and increased tumor burden preceded death or killing of the animal for cause. In contrast, treatment with 8.51 MBq of [177Lu]Lu-ofatumumab dramatically increased median survival (>221 d), permitted weight gain, eliminated detectable tumors, and was curative in 9 of 10 mice. Conclusion: [177Lu]Lu-ofatumumab shows favorable in vitro characteristics, localizes to tumor, and demonstrates curative therapeutic efficacy in a disseminated lymphoma model, showing potential for clinical translation to treat NHL.

Visual Abstract 227Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved 223Ra as its first daughter. There is an ample supply of 227Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Using the CD20-targeting antibody ofatumumab, we evaluated chelation of 227Th4+ for α-particle–emitting and radiotheranostic applications. Methods: We compared 4 bifunctional chelators for thorium radiopharmaceutical preparation: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2-(4-isothicyanatobenzyl)-1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (p-SCN-Bn-HEHA), p-isothiacyanatophenyl-1-hydroxy-2-oxopiperidine-desferrioxamine (DFOcyclo*-p-Phe-NCS), and macrocyclic 1,2-HOPO N-hydroxysuccinimide (L804-NHS). Immunoconstructs were evaluated for yield, purity, and stability in vitro and in vivo. Tumor targeting of the lead 227Th-labeled compound in vivo was performed in CD20-expressing models and compared with a companion 89Zr-labeled PET agent. Results: 227Th-labeled ofatumumab-chelator constructs were synthesized to a radiochemical purity of more than 95%, excepting HEHA. 227Th-HEHA-ofatumumab showed moderate in vitro stability. 227Th-DFOcyclo*-ofatumumab presented excellent 227Th labeling efficiency; however, high liver and spleen uptake was revealed in vivo, indicative of aggregation. 227Th-DOTA-ofatumumab labeled poorly, yielding no more than 5%, with low specific activity (0.08 GBq/g) and modest long-term in vitro stability (<80%). 227Th-L804-ofatumumab coordinated 227Th rapidly and efficiently at high yields, purity, and specific activity (8 GBq/g) and demonstrated extended stability. In vivo tumor targeting confirmed the utility of this chelator, and the diagnostic analog, 89Zr-L804-ofatumumab, showed organ distribution matching that of 227Th to delineate SU-DHL-6 tumors. Conclusion: Commercially available and novel chelators for 227Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for 89Zr/227Th quantitative imaging and α-particle therapy.

Background: The majority of radiopharmaceuticals for use in disease detection and targeted treatment undergo a single radioactive transition (decay) to reach a stable ground state. Complex emitters, which produce a series of daughter radionuclides, are emerging as novel radiopharmaceuticals. The need for validation of chemical and radiopurity with such agents using common quality control instrumentation is an area of active investigation. Here, we demonstrate novel methods to characterize 227Th and 223Ra. Materials and Methods: A radio-TLC scanner and a γ-counter, two common and widely accessible technologies, as well as a solid-state α-particle spectral imaging camera were evaluated for their ability to characterize and distinguish 227Th and 223Ra. We verified these results through purity evaluation of a novel 227Th-labeled protein construct. Results: The γ-counter and α-camera distinguished 227Th from 223Ra, enabling rapid and quantitative determination of radionuclidic purity. The radio-TLC showed limited ability to describe purity, although use under α-particle-specific settings enhanced resolution. All three methods were able to distinguish a pure from impure 227Th-labeled protein. Conclusions: The presented quality control evaluation for 227Th and 223Ra on three different instruments can be applied to both research and clinical settings as new alpha particle therapies are developed.

Immunotherapies that target the CD20 protein expressed on most non-Hodgkin lymphoma cells have improved clinical outcomes, but relapse is common. We prepared 225Ac-labeled anti-CD20 ofatumu-mab and evaluated its in vitro characteristics and therapeutic efficacy in a murine model of disseminated human lymphoma. Methods: 225Ac was chelated by DOTA-ofatumumab, and radiochemical yield, purity, immunoreactivity, stability, and chelate number were deter-mined. In vitro cell killing of CD20-positive, human B-cell lymphoma Raji-Luc cells was assayed. Biodistribution was determined as percentage injected activity per gram (%IA/g) in mice with subcutane-ous Raji-cell tumors (n = 4). [225Ac]Ac-ofatumumab biodistribution in C57BL/6N mice was performed to estimate projected human dosime-try. Therapeutic efficacy was tested in mice with systemically dissemi-nated Raji-Luc cells, tracking survival, bioluminescence, and animal weight for a targeted 200 d, with single-dose therapy initiated 8, 12, or 16 d after cell injection, comparing no treatment, ofatumumab, and low (3.7 kBq/mouse) and high (9.25 kBq/mouse) doses of [225Ac]Ac-IgG and [225Ac]Ac-ofatumumab (n = 8-10/cohort). Results: Radio-chemical yield and purity were 32% & PLUSMN; 9% and more than 95%, respectively. Specific activity was more than 5 MBq/mg. Immunoreac-tivity was preserved, and more than 90% of the 225Ac remained che-lated after 10 d in serum. Raji-Luc cell killing in vitro was significant, specific, and dose-dependent. In tumor-bearing mice, [225Ac]Ac-ofa-tumumab displayed low liver (7 %IA/g) and high tumor (28 %IA/g) uptake. Dosimetry estimates indicated that bone marrow is likely the dose-limiting organ. When therapy was initiated 8 d after cell injection, untreated mice and mice treated with cold ofatumumab or low-or high-dose [225Ac]Ac-IgG showed indistinguishable median survivals of 20-24 d, with extensive cancer-cell burden before death. Low-and high-dose [225Ac]Ac-ofatumumab profoundly (P< 0.05) extended median survival to 190 d and more than 200 d (median not determin-able), with 5 and 9 of 10 mice, respectively, surviving at study termina-tion with no detectable cancer cells. Surviving mice treated with high-dose [225Ac]Ac-ofatumumab showed reduced weight gain ver-sus naive mice. When therapy was initiated 12 d, but not 16 d, after cell injection, high-dose [225Ac]Ac-ofatumumab significantly extended median survival to 40 d but was not curative. Conclusion: In an aggressive disseminated tumor model, [225Ac]Ac-ofatumumab was effective at cancer-cell killing and curative when administered 8 d after cell injection. [225Ac]Ac-ofatumumab has substantial potential for clini- cal translation as a next-generation therapeutic for treatment of patients with non-Hodgkin lymphoma.

BACKGROUND:Many preclinical cancer studies use mice with varied phenotypes to monitor tumor treatment. We compared survival and optical imaging characteristics of strains with varied coat colors harboring luciferase-expressing disseminated lymphoma. RESULTS:Luciferase-expressing lymphoma cells (Raji-luc) were injected via tail vein into severe combined immunodeficient (SCID) and Rag2-IL2rg (R2G2) mice, and survival was tracked. Tumor signals were obtained by imaging ventral and dorsal aspects of mice. Signal attenuation by isolated mouse pelts was measured in vitro. R2G2 mice had decreased survival compared to SCID mice (17 vs. 32 days, p<0.001) despite similar bioluminescence signal when mice were imaged dorsally (p=0.37). However, signal was 17.3-fold higher in R2G2 mice compared to SCID (p<0.001) when imaged ventrally. Isolated dark R2G2 dorsal pelts attenuated signal more than ventral pelts when placed over cells in vitro. CONCLUSIONS:Mouse pelt color and imaging aspect are critical considerations for quantifying bioluminescent tumor signal, and the R2G2 mouse strain may prove useful for preclinical targeted therapy studies.

[223Ra]RaCl2 is the first approved α-particle-emitting therapy and is indicated for treatment of bone metastatic castration-resistant prostate cancer. Approximately half the dose is absorbed into the gastrointestinal tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigated the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Using primary human duodenal organoids (enteroids) as in vitro models of the functional gastrointestinal epithelium, we found that amiloride (epithelial sodium ion channel blocker) and NS-1619 (K+ channel activator) presented significant effects in 223Ra membranal transport. Radioactive drug distribution was evaluated for lead combinations in vivo and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223Ra uptake in vivo from the gut and nearly doubled the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent imaging and radiography was significantly greater than that with single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may readily be implemented as a clinical approach to improve the outcomes of bone-metastatic cancer patients with the benefit of ameliorated tolerability.

1500 Objectives: Immunotherapies that target the CD20 protein present on most non-Hodgkins lymphoma (NHL) cells have greatly improved outcomes. However, as many patients are refractory or undergo relapse, current treatments are inadequate. Our previous work showed that 89Zr-labeled ofatumumab (Ofa), a next-generation, fully human anti-CD20 antibody, effectively targets lymphoma xenografts in SCID mice (Yoon JT, et al. J Nucl Med. 2018;59(8): 1219-1224). Here, we describe characterization of Ofa labeled with lutetium-177, a beta-particle-emitting therapeutic radionuclide, and compare tumor targeting and biodistribution of 177Lu-Ofa with 89Zr-Ofa using an R2G2 immunodeficient mouse model. 177Lu-Ofa has potential advantages over previous beta-particle-based radioimmunotherapies for NHL. Methods: Chx-A-DTPA was conjugated to Ofa at an 8:1 molar ratio. 177Lu was chelated and 177Lu-Ofa purified by size exclusion chromatography. Radiochemical yield and purity were assayed by thin-layer chromatography (TLC). Immunoreactivity was assayed by binding to Raji B-cell lymphoma cells. Stability in human serum was assayed by TLC. Raji-luc tumor-cell killing by 177Lu-Ofa was evaluated by MTS and bioluminescence assays. 89Zr-Ofa preparation, mouse PET/CT imaging, and determination of tissue average standardized uptake values (SUVave), were obtained as described (Yoon et al. 2018). R2G2 mice with subcutaneous Raji xenografts were injected intravenously with 10 µg of 177Lu-Ofa (12.5 µCi, n=5) or 10 µg of 89Zr-Ofa (100 µCi, n=3). Biodistribution of radionuclide was determined 7 d post-injection and are presented as % injected dose/gram (% ID/g). Results: As found in SCID mice, 89Zr-Ofa allowed excellent imaging of CD20-positive tumors in R2G2 mice, with tumor SUVave of over 6, compared to SUVave of about 2 and 1 for bone and muscle, respectively. We obtained 177Lu-Ofa (n=5) with radiochemical yields of 37±7% and over 95% radiochemical purity. Specific activity was over 5 µCi/µg. 177Lu-Ofa immunoreactivity was about 60%, similar to that of 89Zr-Ofa. Serum stability of 177Lu-Ofa was over 90% after 10 d at 37°C. 177Lu-Ofa, but not 177Lu-IgG, showed potent killing in vitro of Raji-luc cells. In tumor-bearing mice, 177Lu-Ofa showed efficient tumor targeting (17±5% ID/g), with tumor levels about two-fold greater than blood levels (9±1% ID/g). Notably,177Lu-Ofa biodistribution was similar to that of 89Zr-Ofa, with a Pearson correlation coefficient (r) value of 0.90 for the thirteen tissues analyzed. Conclusions: 177Lu-Ofa was obtained with high radiochemical purity, immunoreactivity and in vitro stability. 177Lu-Ofa showed high potency in specifically killing CD20-expressing lymphoma cells in vitro. 177Lu-Ofa specifically targeted CD20-expressing lymphoma xenografts, similar to the targeting observed by 89Zr-Ofa, and 177Lu-Ofa vs. 89Zr-Ofa showed highly similar biodistributions in tumor-bearing mice. Together, these results suggest that 177Lu-Ofa has considerable potential for radioimunotherapy of non-Hodgkin lymphoma and that 89Zr-Ofa and 177Lu-Ofa may form a theranostic (imaging and therapeutic) pair for these patients.

Metastatic prostate cancer is incurable, and novel methods to detect the disease earlier and to direct definitive treatment are needed. Molecularly specific tools to localize diagnostic and cytotoxic radionuclide payloads to cancer cells and the surrounding microenvironment are recognized as a critical component of new approaches to combat this disease. The implementation of theranostic approaches to characterize and personalize patient management is beginning to be realized for prostate cancer patients. This review article summarized clinically translated approaches to detect, characterize and treat disease in this rapidly expanding field.

381 Objectives: Immunotherapies that target the CD20 protein present on most non-Hodgkins lymphoma (NHL) cells have greatly improved outcomes. However, as many patients are refractory or undergo relapse, current treatments are inadequate. Our previous work showed that 89Zr-labeled ofatumumab (Ofa), a next-generation, fully human anti-CD20 antibody, effectively targets lymphoma xenografts (Yoon et al, 2018, JNM). Here, we describe characterization of Ofa labeled with Actinium-225 (225Ac-Ofa), which emits 4 cell-killing alpha particles per decay and compare tumor targeting and biodistribution of 225Ac-Ofa with 89Zr-Ofa. 225Ac-Ofa has potential advantages over previous beta-particle-based radioimmunotherapies for NHL. Methods: DOTA was conjugated to Ofa at an 8:1 molar ratio. 225Ac was chelated and 225Ac-Ofa purified by size exclusion chromatography (SEC). Radiochemical yield was assayed by iTLC and radiochemical purity by iTLC and fast protein liquid chromatography (FPLC). Immunoreactivity was assayed by binding to Raji B-cell lymphoma cells. Stability in human serum was assayed by iTLC. Raji-luc tumor-cell killing by 225Ac-Ofa was evaluated by MTS and bioluminescence assays. 89Zr-Ofa was prepared as described (Yoon et al.). Mice with subcutaneous Raji xenografts were injected IV with 300 nCi (10 µg) 225Ac-Ofa (n=4), 100 µCi (10 µg) 89Zr-Ofa (n=3) or 300 nCi free 225Ac (n=5). Naive mice were injected IV with 225Ac-Ofa (n=4) or 89Zr-Ofa (n=4). Biodistribution in 20 organs/tissues were determined 7 d post-injection as % injected dose/gram. Results: We obtained 225Ac-Ofa (n=5) with radiochemical yields of 36±6% and >98% radiochemical purity. FPLC confirmed >98% of the 225Ac was bound to Ofa. Specific activity was >100 nCi/µg. 225Ac-Ofa immunoreactivity was >65%, similar to 89Zr-Ofa. Serum stability was 70-90% after 10 d at 37°C (n=3). 225Ac-Ofa showed potent and dose-dependent killing of Raji-luc cells at concentrations from 10-100 nCi/ml vs. 225Ac-IgG or unlabeled Ofa (p 0.05) to 89Zr-Ofa (43±13% ID/g). Biodistribution was also not statistically different between 225Ac-Ofa and 89Zr-Ofa in liver (31±6 vs. 21±2% ID/g), blood (10±3 vs. 9±2% ID/g), spleen (31±6 vs. 21±3 % ID/g), bone marrow (7±2 vs. 8±2 % ID/g) or other issues analyzed. In tumor-bearing mice, free 225Ac accumulated to high levels in the liver (64±19% ID/g), significantly (p 0.05) localizations to blood (16±1 vs. 13±2% ID/g), liver (10±2 vs. 9±1% ID/g), spleen (34±6 vs. 22±7% ID/g), bone marrow (15±8 vs. 10±4% ID/g) and other tissues. Conclusions: 225Ac-Ofa was obtained with high radiochemical purity, immunoreactivity and in vitro stability. 225Ac-Ofa showed high potency in specifically killing CD20-expressing lymphoma cells in vitro. 225Ac-Ofa specifically targeted CD20-expressing lymphoma xenografts, similar to the targeting observed by 89Zr-Ofa, and 225Ac-Ofa vs. 89Zr-Ofa showed highly similar biodistributions to non-target organs/tissues. Together, these results suggest 225Ac-Ofa has considerable potential for radioimunotherapy of non-Hodgkin lymphoma and that 89Zr-Ofa and 225Ac-Ofa may form a theranostic (imaging and therapeutic) pair for these patients.

1000 Objectives: Immune checkpoint blockade (ICB) therapies are often effective for cancer treatment. Atezolizumab (Atz), a fully humanized anti-PD-L1 monoclonal antibody (mAb), is approved for ICB therapy for lung and bladder cancer. Despite dramatic success with Atz in some patients, we cannot yet reliably predict who will respond positively to Atz treatment, but favorable response appears related to PD-L1 levels in the tumor microenvironment. Immunohistochemical assessment of tumor PD-L1 levels is invasive and requires biopsy, which is often impractical, particularly in metastatic disease or for assessing disease over time. With the objective of non-invasive tumor PD-L1 assessment, we generated Atz labeled with the PET isotope, 89Zr (89Zr-Atz), and evaluated its ability to target xenograft tumors in mice that express human PD-L1 (hPD-L1). METHODS: Atz was conjugated with deferoxamine (DFO) at 10:1, 5:1, and 2:1 DFO:mAb ratios and 89Zr chelated. Radiochemical yield, purity, stability and aggregation were assayed by instant thin-layer chromatography (iTLC) and FPLC. 89Zr-Atz immunoreactivity was quantified by cell binding and Kds of Atz and Atz-DFO determined by enzyme-linked immunosorbent assays (ELISA) and of 89Zr-Atz by cell binding. Binding to mouse PD-L1 (mPD-L1) was assayed by flow cytometry and ELISA. SCID mice (n≥4) with bilateral shoulder xenografts of Cho and Cho-PD-L1 cells were injected with 3.7 MBq (~11 µg) of 89Zr-Atz without or with cold Atz (10, 50, 250 or 1250 µg) or 250 µg cold IgG. 89Zr-IgG alone was also injected. Biodistribution was determined 7 days post-injection (p.i.) and standardized uptake values (SUVmax and SUVave) determined from PET/CT images acquired at days 1, 3 and 7 p.i. RESULTS: We obtained 89Zr-Atz radiochemical yields of >260 MBq/mg, with >95% purity and >90% immunoreactivity. Atz, Atz-DFO and 89Zr-Atz exhibited similar hPD-L1 Kds of 95% 89Zr remained bound after 7 days in serum. Aggregation of Atz-DFO and 89Zr-Atz were less than 10% and Atz bound mPD-L1. 89Zr-IgG did not specifically target PD-L1 expressing tumors, as assayed by biodistribution (Table 1) or SUV analysis. Similarly, specific targeting was not observed with 89Zr-Atz alone, with co-injection of cold IgG, or with co-injection of 10, 50, or 1250 µg cold IgG. Blood levels of 89Zr-Atz were low in the absence of co-injected cold Atz, indicating saturable binding to mPD-L1. In contrast, co-injection of 250 µg of cold Atz resulted in specific 89Zr-Atz targeting of PD-L1 expressing tumor cells by biodistribution (Table 1, [asterisk]p<0.05) and SUV analyses (e.g., day 3 SUVave Cho 1.2±0.3 vs. Cho-PD-L1 2.6±0.3, p<0.05). CONCLUSIONS: 89Zr-Atz-DFO was generated with high immunoreactivity and specific activity. Atz bound to both mouse and human PD-L1, and 89Zr-Atz recognized mouse tissues expressing PD-L1. The ability of 89Zr-Atz to specifically target tumor cells expressing PD-L1 was highly dependent on Atz protein mass. These results suggest that the amount of co-injected cold Atz with 89Zr-Atz will be critical for maximal detection of PD-L1 in the tumor microenvironment in future clinical trials. We thank Sridhar Nimmagadda for the Cho-PD-L1 cell line.

Radioimmunotherapies with monoclonal antibodies to the B-lymphocyte antigen 20 (CD20) are effective treatments for B-cell lymphomas, but U.S. Food and Drug Administration–approved radioimmunotherapies exclusively use radiolabeled murine antibodies, potentially limiting redosing. The Food and Drug Administration recently approved 2 unlabeled anti-CD20 monoclonal antibodies, obinutuzumab and ofatumumab, termed next generation as they are humanized (obinutuzumab) or fully human (ofatumumab), thus potentially allowing a greater potential for redosing than with previous-generation anti-CD20 antibodies, including rituximab (chimeric) and tositumomab (murine), which contain more murine peptide sequences. We prepared 89Zr-ofatumumab and 89Zr-obinituzumab and assessed their tumor targeting by PET/CT imaging and their biodistribution in a preclinical mouse model with CD20 xenografts to determine whether these antibodies have potential as theranostics or for radioimmunotherapy. Methods: Obinutuzumab, ofatumumab, rituximab, tositumomab, and human IgG (as control) were radiolabeled with 89Zr. Raji Burkitt lymphoma xenografts were established in severe combined immunodeficient mice. Mice with palpable tumors (n = 4–9) were injected with 89Zr-obinutuzumab, 89Zr-ofatumumab, 89Zr-rituximab, 89Zr-tositumomab, or 89Zr-IgG, with small-animal PET/CT images acquired at 1, 3, and 7 d after injection, and then sacrificed for biodistribution analyses. Results: At 1, 3, and 7 d after injection, all anti-CD20 antibodies showed clear tumor uptake on PET/CT, with minimal tumor uptake of IgG. Biodistribution data showed significantly (P < 0.005) higher tumor uptake for obinutuzumab (41.4 ± 7.6 percentage injected dose [%ID]/g), ofatumumab (32.6 ± 17.5 %ID/g), rituximab (28.6 ± 7.6 %ID/g), and tositumomab (28.0 ± 6.5 %ID/g) than IgG (7.2 ± 1.2 %ID/g). Tositumomab had much higher splenic uptake (186.4 ± 49.7 %ID/g, P < 0.001) than the other antibodies. Conclusion:89Zr-labeled obinutuzumab and ofatumumab localized to tumor as well as or better than labeled rituximab and tositumomab, 2 monoclonal antibodies that have been used previously in B-cell lymphoma radioimmunotherapy, and both obinutuzumab and ofatumumab have the potential for repeated dosing.

INTRODUCTION:There is a need for prophylaxis to reduce placental-associated intrauterine growth restriction (IUGR). Pomegranate juice (PJ) is replete with phytochemicals having biological effects at non-pharmacological concentrations. We test the hypothesis that exposure of pregnant mice to hypoxia late in gestation induces cellular stress in the placenta, which can be ameliorated by antecedent maternal consumption of PJ.MATERIALS AND METHODS:We exposed pregnant mice to 12% or 21% oxygen, with food ad libitum or restricted, and with consumption of PJ or glucose between 12.5 and 18.5 days post conception (dpc). We examined the outcomes of the nine groups (n = 10) at 18.5 dpc, quantifying fetal and placental weights and placental labyrinthine and junctional zone depths and areas. We assayed cellular stress by expression of Hsp90 and apoptosis by TUNEL staining and expression of cleaved caspase 3.RESULTS:Maternal exposure to 12% oxygen or food restriction in 21% oxygen, induced IUGR, compared to control. The labyrinth to junctional zone ratio was lower in hypoxic ad libitum, compared to normoxic food-restricted, placentas. Antenatal PJ prior to and during hypoxic exposure significantly improved fetal growth, reduced Hsp90 expression, and limited apoptosis in the labyrinth, while enhancing junctional zone apoptosis.DISCUSSION:Maternal exposure to hypoxia induces IUGR, cell stress, and apoptosis in mouse placentas. The labyrinth and junctional zone of the mouse placenta are differentially sensitive to FiO2 and to PJ. PJ offers benefits in the prophylaxis of IUGR in the mouse, but PJ effects on the junctional zone require further study.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1β and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

We assessed the response of primary cultures of placental villous mononucleated trophoblasts and multinucleated syncytiotrophoblast to calcitriol, the most biologically active form of vitamin D. Whole-genome microarray data showed that calcitriol modulates the expression of many genes in trophoblasts within 6 hours of exposure and RT-qPCR revealed similar responses in cytotrophoblasts, syncytiotrophoblasts and villous explants. Both cytotrophoblasts and syncytiotrophoblasts expressed genes for the vitamin D receptor, for LRP2 and CUBN that mediate internalization of calcidiol, for CYP27B1 that encodes the enzyme that converts calcidiol into active calcitriol, and for CYP24A1 that encodes the enzyme that modifies calcitriol and calcidiol to inactive calcitetrol. Notably, we found an inverse effect of calcitriol on expression of CD14 and CD180/RP105, proteins that differentially regulate toll-like receptor 4-mediated immune responses. Supported by gene ontology analysis, we tested the hypothesis that CD14 and CD180 modulate the inflammatory response of syncytiotrophoblast to bacterial lipopolysaccharide (LPS). These cells showed a robust response to a wide range of LPS concentrations, with induction of active NF-κB and increased secretion of IL-6 and IL-8. SiRNA-mediated knockdown of CD14 reduced the secretion of IL-6 and IL-8 in response to LPS. Collectively, our data showed that calcitriol has a rapid and widespread effect on villous trophoblast gene expression in general, and a specific effect on the innate immune response by syncytiotrophoblast.