The TLR2/TLR6 ligand FSL-1 mitigates radiation-induced hematopoietic injury in mice and nonhuman primates.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Animal Model To Study Klebsiella pneumoniae Gastrointestinal Colonization and Host-to-Host Transmission.

An important yet poorly understood facet of the life cycle of a successful pathogen is host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae, a Gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat, as K. pneumoniae has become multidrug resistant. Epidemiological studies suggest that K. pneumoniae host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Here, we describe a murine model that can be utilized to study mucosal (oropharynx and gastrointestinal [GI]) colonization, shedding within feces, and transmission of K. pneumoniae through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we showed that K. pneumoniae can asymptomatically colonize the GI tract in immunocompetent mice and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent K. pneumoniae isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, K. pneumoniae carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient supershedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors toward K. pneumoniae dissemination.

Endometrium and endometriosis tissue mitochondrial energy metabolism in a nonhuman primate model.

BACKGROUND: Endometriosis is the growth of uterine lining (endometrium) outside of the uterus. In other chronic inflammatory diseases, mitochondrial dysfunction is suspected of playing a role in disease pathogenesis. However, little is known about endometriosis mitochondrial function or its effects on tissue metabolism. The objectives of this study were to analyze mitochondrial function in nonhuman primate (NHP) endometrium and endometriosis tissue and to identify the metabolic features of these tissues that may contribute to disease. METHODS: Mitochondrial function in endometriosis tissue and endometrium was measured using mitochondrial respirometry analysis to determine if changes in oxidative phosphorylation exist in endometrium and endometriosis tissue compared to control endometrium from clinically healthy NHPs. Targeted metabolomics and multidimensional statistical analysis were applied to quantify key metabolites in energy and amino acid biosynthesis pathways. RESULTS: Mitochondrial respirometry assays showed endometrium from NHPs with endometriosis had reduced complex II-mediated oxygen consumption rates (OCR) across all energy states (basal, p = 0.01; state 3, p = 0.02; state 3u, p = 0.04; state 4o, p = 0.008) and endometriosis tissue had reduced state 3, complex I-mediated OCR (p = 0.02) and respiratory control rates (p = 0.01) compared to normal endometrium. Targeted metabolomics performed on tissue revealed carnitine (p = 0.001), creatine phosphate (p = 0.01), NADH (p = 0.0001), FAD (p = 0.001), tryptophan (p = 0.0009), and malic acid (p = 0.005) were decreased in endometriosis tissue compared to normal endometrium samples. FAD (p = 0.004), tryptophan (p = 0.0004) and malic acid (p = 0.03) were significantly decreased in endometrium from NHPs with endometriosis compared to normal endometrium. Significant metabolites identified in endometriosis and endometrium samples from animals with endometriosis were part of amino acid biosynthesis or energy metabolism pathways. CONCLUSIONS: Here, endometrial mitochondrial energy production and metabolism were decreased in endometrium and endometriosis tissue. Decreased mitochondrial energy production may be due to oxidative stress-induced damage to mitochondrial DNA or membranes, a shift in cell metabolism, or decreased energy substrate; however, the exact cause remains unknown. Additional research is needed to determine the implications of reduced mitochondrial energy production and metabolism on endometriosis and endometrium.

Mechanisms of human erythrocytic bioactivation of nitrite.

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO2 are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.

Specific HDAC6 inhibition by ACY-738 reduces SLE pathogenesis in NZB/W mice.

We sought to determine if a selective HDAC6 inhibitor (ACY-738) decreases disease in NZB/W mice. From 22 to 38weeks-of-age, mice were injected intraperitoneally with 5 or 20mg/kg of ACY-738, or vehicle control. Body weight and proteinuria were measured every 2weeks, while sera anti-dsDNA, Ig isotypes, and cytokine levels were measured every 4weeks. Kidney disease was determined by evaluation of sera, urine, immune complex deposition, and renal pathology. Flow cytometric analysis assessed thymic, splenic, bone marrow, and peripheral lymphocyte differentiation patterns. Our results showed HDAC6 inhibition decreased SLE disease by inhibiting immune complex-mediated glomerulonephritis, sera anti-dsDNA levels, and inflammatory cytokine production and increasing splenic Treg cells. Inhibition of HDAC6 increased the percentage of cells in the early-stage developmental fractions of both pro- and pre-B cells. These results suggest that specific HDAC6 inhibition may be able to decrease SLE disease by altering aberrant T and B cell differentiation.

DMP1ß, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo-insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1ß and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1ß expression. RNA-seq analyses of a publicly available database showed significantly increased DMP1ß mRNA in 43-55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1ß protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1ß overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1ß may have a biological function. Indeed, DMP1ß increased proliferation of non-tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1ß's role in vivo, we established MMTV-DMP1ß transgenic mouse lines. DMP1ß overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7-18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up-regulating the tumour-promoting DMP1ß isoform.

Downregulation of apelin in the human placental chorionic villi from preeclamptic pregnancies.

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36-38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7±3.4 vs. 72.3±9.8 fmol/mg protein; n=20-22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0±23.0 vs. 485.3±24.8 pmol·mg(-1)·min(-1); n=18-22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi (P<0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II.

Assessment of Multiplate platelet aggregometry using citrate, heparin or hirudin in Rhesus macaques.

Electrical impedance aggregometry (EIA) has gained popularity in clinical and research applications. Nonhuman primates are used to study disease and drug-related mechanisms that affect hemostasis, therefore establishing normal EIA parameters are necessary. The anticoagulants sodium heparin, hirudin and sodium citrate and three agonists, ADP, ASPI, and collagen were evaluated. Whole blood from 12 adult male rhesus macaques was collected to evaluate anticoagulants, sodium heparin, hirudin and sodium citrate using three agonists (ADP, ASPI and collagen), on the Multiplate® 5.0 Analyzer. Platelet function was reported for three parameters: Area under the curve (AUC), aggregation, and aggregation velocity. There was a significant difference in mean AUC between citrate and heparin samples, and citrate and hirudin samples regardless of the agonist used. There was no difference in AUC between heparin and hirudin. ADP-activated samples showed an increase in impedance with hirudin samples compared to citrate. Furthermore, heparin and hirudin out-perform citrate as the anticoagulant for EIA in the macaque. Finally, this study demonstrates the utility of the Multiplate® system in this model and provides important insight into anticoagulant choice when using EIA.

Class I and II histone deacetylase inhibition by ITF2357 reduces SLE pathogenesis in vivo.

We sought to determine if a specific class I and II HDAC inhibitor (ITF2357) was able to decrease disease in lupus-prone NZB/W mice through regulation of T cell profiles. From 22 to 38 weeks-of-age, NZB/W and non-lupus NZW mice were treated with ITF2357 (5 mg/kg or 10 mg/kg), or vehicle control. Body weight and proteinuria were measured every 2 weeks, while sera anti-dsDNA and cytokine levels were measured every 4 weeks. Kidney disease was determined by sera IgG levels, immune complex deposition, and renal pathology. T lymphocyte profiles were assessed using flow cytometric analyses. Our results showed that NZB/W mice treated with the 10 mg/kgof ITF2357 had decreased renal disease and inflammatory cytokines in the sera. Treatment with ITF2357 decreased the Th17 phenotype while increasing the percentage of Tregs as well as Foxp3 acetylation. These results suggest that specific HDAC inhibition may decrease disease by altering T cell differentiation and acetylation.

Systemic immune response to a CD40 agonist antibody in nonhuman primates.

The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is broadly expressed by immune cells including B cells, dendritic cells, and monocytes, as well as other normal cells and some malignant cells. CD40 is constitutively expressed on antigen-presenting cells, and ligation promotes functional maturation, leading to an increase in antigen presentation and cytokine production, and a subsequent increase in the activation of antigen-specific T cells. It is postulated that CD40 agonists can mediate both T cell-dependent and T cell-independent immune mechanisms of tumor regression in mice and patients. In addition, it is believed that CD40 activation also promotes apoptotic death of tumor cells and that the presence of the molecule on the surface of cancer cells is an important factor in the generation of tumor-specific T cell responses that contribute to tumor cell elimination. Notably, CD40 agonistic therapies were evaluated in patients with solid tumors and hematologic malignancies with reported success as a single agent. Preclinical studies have shown that subcutaneous administration of CD40 agonistic antibodies reduces systemic toxicity and elicits a stronger and localized pharmacodynamic response. Two independent studies in cynomolgus macaque (Macaca fascicularis) were performed to further evaluate potentially immunotoxicological effects associated with drug-induced adverse events seen in human subjects. Studies conducted in monkeys showed that when selicrelumab is administered at doses currently used in clinical trial patients, via subcutaneous injection, it is safe and effective at stimulating a systemic immune response.

Long-Term Immunological Consequences of Radiation Exposure in a Diverse Cohort of Rhesus Macaques.

PURPOSE: The aim of this study was to develop an improved understanding of the delayed immunologic effects of acute total body irradiation (TBI) using a diverse cohort of nonhuman primates as a model for an irradiated human population. METHODS AND MATERIALS: Immune recovery was evaluated in 221 rhesus macaques either left unirradiated (n = 36) or previously irradiated (n = 185) at 1.1 to 8.5 Gy TBI (median, 6.5 Gy) when aged 2.1 to 15.5 years (median, 4.2 years). Blood was drawn annually for up to 5 years total between 0.5 and 14.3 years after exposure. Blood was analyzed by complete blood count, immunophenotyping of monocytes, dendritic cells (DC) and lymphocytes by flow cytometry, and signal joint T-cell receptor exclusion circle quantification in isolated peripheral blood CD4 and CD8 T cells. Animals were categorized by age, irradiation status, and time since irradiation. Sex-adjusted means of immune metrics were evaluated by generalized estimating equation models to identify cell populations altered by TBI. RESULTS: Overall, the differences between irradiated and nonirradiated animals were subtle and largely restricted to younger animals and select cell populations. Subsets of monocytes, DC, T cells, and B cells showed significant interaction effects between radiation dose and age after adjustment for sex. Irradiation at a young age caused transient increases in the percentage of peripheral blood myeloid DC and dose-dependent changes in monocyte balance for at least 5 years after TBI. TBI also led to a sustained decrease in the percentage of circulating memory B cells. Young irradiated animals exhibited statistically significant and prolonged disruption of the naïve/effector memory/central memory CD4 and CD8 T-cell equilibrium and exhibited a dose-dependent increase in thymopoiesis for 2 to 3 years after exposure. CONCLUSIONS: This study indicates TBI subtly but significantly alters the circulating proportions of cellular mediators of adaptive immune memory for several years after irradiation, especially in macaques under 5 years of age and those receiving a high dose of radiation.

Delayed effects of radiation in adipose tissue reflect progenitor damage and not cellular senescence.

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAß-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-ßgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

Total-Body Irradiation Is Associated With Increased Incidence of Mesenchymal Neoplasia in a Radiation Late Effects Cohort of Rhesus Macaques (Macaca mulatta).

PURPOSE: Cancer is a severe delayed effect of acute radiation exposure. Total-body irradiation has been associated with an increased risk of solid cancer and leukemia in Japanese atomic bomb survivors, and secondary malignancies, such as sarcoma, are a serious consequence of cancer radiation therapy. The radiation late effects cohort (RLEC) of rhesus macaques (Macaca mulatta) is a unique resource of more than 200 animals for studying the long-term consequences of total-body irradiation in an animal model that closely resembles humans at the genetic and physiologic levels. METHODS AND MATERIALS: Using clinical records, clinical imaging, histopathology, and immunohistochemistry, this retrospective study characterized the incidence of neoplasia in the RLEC. RESULTS: Since 2007, 61 neoplasms in 44 of 239 irradiated animals were documented (18.4% of the irradiated population). Only 1 neoplasm was diagnosed among the 51 nonirradiated controls of the RLEC (2.0%). The most common malignancies in the RLEC were sarcomas (38.3% of diagnoses), which are rare neoplasms in nonirradiated macaques. The most common sarcomas included malignant nerve sheath tumors and malignant glomus tumors. Carcinomas were less common (19.7% of diagnoses), and consisted primarily of renal cell and hepatocellular carcinomas. Neoplasia occurred in most major body systems, with the skin and subcutis being the most common site (40%). RNA analysis showed similarities in transcriptional profiles between RLEC and human malignant nerve sheath tumors. CONCLUSIONS: This study indicates that total-body irradiation is associated with an increased incidence of neoplasia years following irradiation, at more than double the incidence described in aging, nonirradiated animals, and promotes tumor histotypes that are rarely observed in nonirradiated, aging rhesus macaques.

Epizootic Yersinia enterocolitica in captive African green monkeys (Chlorocebus aethiops sabaeus).

Yersinia enterocolitica is a Gram-negative bacterium that typical results in enterocolitis in humans and poses significant worldwide risks to public health. An outbreak of yersiniosis in the Vervet/African green monkey colony at the WFSM during the winter of 2015-2016 accounted for widespread systemic infection with high morbidity and mortality. Most of the cases had extensive necrosis with suppuration and large colonies of bacilli in the large bowel and associated lymph nodes; however, the small intestine, stomach, and other organs were also regularly affected. Positive cultures of Yersinia enterocolitica were recovered from affected tissues in 20 of the 23 cases. Carrier animals in the colony were suspected as the source of the infection because many clinically normal animals were culture-positive during and after the outbreak. In this study, we describe the gross and histology findings and immune cell profiles in different organs of affected animals. We found increased numbers of myeloid-derived phagocytes and CD11C-positive antigen-presenting cells and fewer adaptive T and B lymphocytes, suggesting an immunocompromised state in these animals. The pathogen-mediated microenvironment may have contributed to the immunosuppression and rapid spread of the infection in the vervets. Further studies in vervets could provide a better understanding of Yersinia-mediated pathogenesis and immunosuppression, which could be fundamental to understanding chronic and systemic inflammatory diseases in humans.

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen's Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (<0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Improved Antitumor Activity of the Fluoropyrimidine Polymer CF10 in Preclinical Colorectal Cancer Models through Distinct Mechanistic and Pharmacologic Properties.

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU's use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo, CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10's antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer.

Tetravalent dengue DNA vaccine is not immunogenic when delivered by retrograde infusion into salivary glands.

INTRODUCTION AND BACKGROUND: A tetravalent DNA vaccine for Dengue virus is under development but has not yet achieved optimal immunogenicity. Salivary glands vaccination has been reported efficacious in rodents and dogs. We report on a pilot study testing the salivary gland as a platform for a Dengue DNA vaccine in a non-human primate model. MATERIALS AND METHODS: Four cynomolgus macaques were used in this study. Each macaque was pre-medicated with atropine and sedated with ketamine. Stensen's duct papilla was cannulated with a P10 polyethylene tube, linked to a 500ul syringe. On the first two infusions, all macaques were infused with 300ul of TVDV mixed with 2 mg of zinc. For the 3rd infusion, to increase transfection into salivary tissue, two animals received 100uL TVDV mixed with 400uL polyethylenimine 1µg/ml (PEI) and the other two animals received 500uL TVDV with zinc. Antibody titers were assessed 4 weeks following the second and third infusion. RESULTS AND CONCLUSIONS: SGRI through Stensen's duct is a well-tolerated, simple and easy to reproduce procedure. TVDV infused into macaques salivary glands elicited a significantly weaker antibody response than with different delivery methods.

Transcriptional Profiling of Non-Human Primate Lymphoid Organ Responses to Total-Body Irradiation.

The global threat of exposure to radiation and its subsequent outcomes require the development of effective strategies to mitigate immune cell injury. In this study we explored transcriptional and immunophenotypic characteristics of lymphoid organs of a non-human primate model after total-body irradiation (TBI). Fifteen middle-aged adult, ovariectomized, female cynomolgus macaques received a single dose of 0, 2 or 5 Gy gamma radiation. Thymus, spleen and lymph node from three controls and 2 Gy (n = 2) and 5 Gy (n = 2) exposed animals were assessed for molecular responses to TBI through microarray-based transcriptional profiling at day 5 postirradiation, and cellular changes through immunohistochemical (IHC) characterization of markers for B and T lymphocytes and macrophages across all 15 animals at time points up to 6 months postirradiation. Irradiated macaques developed acute hematopoietic syndrome. Analysis of array data at day 5 postirradiation identified transcripts with ≥2-fold difference from control and a false discovery rate (FDR) of Padj < 0.05 in lymph node (n = 666), spleen (n = 493) and thymus (n=3,014). Increasing stringency of the FDR to P < 0.001 reduced the number of genes to 71 for spleen and 379 for thymus. IHC and gene expression data demonstrated that irradiated animals had reduced numbers of T and B lymphocytes along with relative elevations of macrophages. Transcriptional analysis revealed unique patterns in primary and secondary lymphoid organs of cynomolgus macaques. Among the many differentially regulated transcripts, upregulation of noncoding RNAs [MIR34A for spleen and thymus and NEAT1 (NCRNA00084) for thymus] showed potential as biomarkers of radiation injury and targets for mitigating the effects of radiation-induced hematopoietic syndrome-impaired lymphoid reconstitution.