Primate Cells Research Articles

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

The mechanism that explains the association of APOL1 variants with nondiabetic kidney diseases in African Americans remains unclear. Kidney disease risk is inherited as a recessive trait, and many studies investigating the intracellular function of APOL1 have indicated the APOL1 variants G1 and G2 are associated with cytotoxicity. Whether cytotoxicity results from the absence of a protective effect conferred by the G0 allele or is induced by a deleterious effect of variant allele expression has not be conclusively established. A central issue hampering basic biology studies is the lack of model systems that authentically replicate APOL1 expression patterns. APOL1 is present in humans and a few other primates and appears to have important functions in the kidney, as the kidney is the primary target for disease associated with the genetic variance. There have been no studies to date assessing the function of untagged APOL1 protein under native expression in human or primate kidney cells, and no studies have examined the heterozygous state, a disease-free condition in humans. A second major issue is the chronic kidney disease (CKD)-associated APOL1 variants are conditional mutations, where the disease-inducing function is only evident under the appropriate environmental stimulus. In addition, it is possible there may be more than one mechanism of pathogenesis that is dependent on the nature of the stressor or other genetic variabilities. Studies addressing the function of APOL1 and how the CKD-associated APOL1 variants cause kidney disease are challenging and remain to be fully investigated under conditions that faithfully model known human genetics and physiology.

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission

A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (−29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

Neuroprotective efficacy of P7C3 compounds in primate hippocampus

There is a critical need for translating basic science discoveries into new therapeutics for patients suffering from difficult to treat neuropsychiatric and neurodegenerative conditions. Previously, a target-agnostic in vivo screen in mice identified P7C3 aminopropyl carbazole as capable of enhancing the net magnitude of postnatal neurogenesis by protecting young neurons from death. Subsequently, neuroprotective efficacy of P7C3 compounds in a broad spectrum of preclinical rodent models has also been observed. An important next step in translating this work to patients is to determine whether P7C3 compounds exhibit similar efficacy in primates. Adult male rhesus monkeys received daily oral P7C3-A20 or vehicle for 38 weeks. During weeks 2–11, monkeys received weekly injection of 5′-bromo-2-deoxyuridine (BrdU) to label newborn cells, the majority of which would normally die over the following 27 weeks. BrdU+ cells were quantified using unbiased stereology. Separately in mice, the proneurogenic efficacy of P7C3-A20 was compared to that of NSI-189, a proneurogenic drug currently in clinical trials for patients with major depression. Orally-administered P7C3-A20 provided sustained plasma exposure, was well-tolerated, and elevated the survival of hippocampal BrdU+ cells in nonhuman primates without adverse central or peripheral tissue effects. In mice, NSI-189 was shown to be pro-proliferative, and P7C3-A20 elevated the net magnitude of hippocampal neurogenesis to a greater degree than NSI-189 through its distinct mechanism of promoting neuronal survival. This pilot study provides evidence that P7C3-A20 safely protects neurons in nonhuman primates, suggesting that the neuroprotective efficacy of P7C3 compounds is likely to translate to humans as well.

A novel antiviral inhibits Zika virus infection while increasing intracellular glutathione biosynthesis in distinct cell culture models.

We investigated the effects of a specific free-form amino acids formulation on Zika virus replication in two different cell culture model systems, one representative of humans and the other of Old World primates from whom Zika virus was first isolated. Here we present data demonstrating that the formulation of the specific free-form amino acid (FFAAP), comprising cystine, glycine, and a glutamate source, along with a minute concentration of selenium inhibited Zika virus replication by up to 90% with an ED90 (effective dose at which 90% of a dose of Zika virus was inhibited) of 2.5 mM in human cells and 4 mM Vero cells. The ED90 concentration of precursors was innocuous for uninfected cells, but resulted in reduced Zika virus replication by up to 90% at 2–5 mM concentrations in nonhuman primate cells and at 1–3 mM concentration in human placental cells.Two important observations were forthcoming: 1) Zika virus production was decreased by up to 90% in Vero and JEG-3 cells treated with FFAAP (ED90 4.0 mM, and 2.5 mM, respectively) throughout 48–72 h of post infection (hpi) compared to untreated infected cells and 2) Zika virus requires intracellular glutathione for replication in human placental cells, while showing enhanced replication in Vero cells with no glutathione. Relative increases in intracellular glutathione biosynthesis followed FFAAP treatment but blocking intracellular biosynthesis of glutathione in human cells resulted in virus inhibition in human placental cells. The blockade of biosynthesis actually increased Zika virus replication in Vero cells. These findings identify an efficacious inhibitor, FFAAP, of Zika virus replication in both human and nonhuman primate cells, while providing novel insight into the different roles of intracellular glutathione in Zika virus replication.

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes.

Dengue virus (DENV), an arthropod-borne (“arbovirus”) virus, causes a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome and proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Mosquitoes and primates show taxonomic group-specific intolerance to certain codon pairs when expressing their genes by translation. This is called “codon pair bias”. By necessity, dengue viruses evolved to delicately balance this fundamental difference in their open reading frames (ORFs). We have undone the evolutionarily conserved genomic balance in the DENV2 ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV2 raised concerns of ‘gain-of-function,’ namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell lines and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control DENV2’s host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

The Virology of Taterapox Virus In Vitro.

Taterapox virus (TATV) is phylogenetically the closest related virus to variola—the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

Single-cell RNA-sequencing reveals the existence of naive and primed pluripotency in pre-implantation rhesus monkey embryos.

Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage. Importantly, we uncovered the existence of distinct pluripotent states in monkey pre-implantation embryos. At the early- and middle-blastocyst stages, the epiblast cells have the transcriptome features of naive pluripotency, whereas they display a continuum of primed pluripotency characteristics at the late and hatched blastocyst stages. Moreover, we identified potential regulators that might play roles in the transition from naive to primed pluripotency. Thus, our study suggests the transient existence of naive pluripotency in primates and proposes an ideal time window for derivation of primate embryonic stem cells with naive pluripotency.

Primate stem cells: bridge the translation from basic research to clinic application.

A growing body of literature has shown that stem cells are very effective for the treatment of degenerative diseases in rodents but these exciting results have not translated to clinical practice. The difference results from the divergence in genetic, metabolic, and physiological phenotypes between rodents and humans. The high degree of similarity between non-human primates (NHPs) and humans provides the most accurate models for preclinical studies of stem cell therapy. Using a NHP model to understand the following key issues, which cannot be addressed in humans or rodents, will be helpful for extending stem cell applications in the basic science and the clinic. These issues include pluripotency of primate stem cells, the safety and efficiency of stem cell therapy, and transplantation procedures of stem cells suitable for clinical translation. Here we review studies of the above issues in NHPs and current challenges of stem cell applications in both basic science and clinical therapies. We propose that the use of NHP models, in particular combining the serial production and transplantation procedures of stem cells is the most useful for preclinical studies designed to overcome these challenges.

Progressive lentivirus infection induces natural killer cell receptor-expressing B cells in the gastrointestinal tract.

Recently, a seemingly novel innate immune cell subset bearing features of natural killer and B cells was identified in mice. So-called NKB cells appear as first responders to infections, but whether this cell population is truly novel or is in fact a subpopulation of B cells and exists in higher primates remains unclear. The objective of this study was to identify NKB cells in primates and study the impact of HIV/SIV infections. NKB cells were quantified in both naive and lentivirus infected rhesus macaques and humans by excluding lineage markers (CD3, CD127) and positive Boolean gating for CD20, NKG2A/C and/or NKp46. Additional phenotypic measures were conducted by RNA-probe and traditional flow cytometry. Circulating cytotoxic NKB cells were found at similar frequencies in humans and rhesus macaques (range, 0.01-0.2% of total lymphocytes). NKB cells were notably enriched in spleen (median, 0.4% of lymphocytes), but were otherwise systemically distributed in tonsil, lymph nodes, colon, and jejunum. Expression of immunoglobulin was highly variable, but heavily favoured IgM and IgA rather than IgG. Interestingly, NKB cell frequencies expanded in PBMC and colon during SIV infection, as did IgG expression, but were generally unaltered in HIV-infected humans. These results suggest a cell type expressing both natural killer and B-cell features exists in rhesus macaques and humans and are perturbed by HIV/SIV infection. The full functional niche remains unknown, but the unique phenotype and systemic distribution could make NKB cells unique targets for immunotherapeutics or vaccine strategies.

Murine and Non-Human Primate Dendritic Cell Targeting Nanoparticles for in Vivo Generation of Regulatory T-Cells.

Porous silicon nanoparticles (pSiNP), modified to target dendritic cells (DC), provide an alternate strategy for the delivery of immunosuppressive drugs. Here, we aimed to develop a DC-targeting pSiNP displaying c-type lectin, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), and CD11c monoclonal antibodies. The in vivo tracking of these fluorescent DC-targeting nanoparticles was assessed in both C57BL/6 mice and common marmosets ( Callithrix jacchus) by intravenous injection (20 mg/kg). Rapamycin and ovalbumin (OVA)323-339 peptide loaded pSiNP were employed to evaluate their ability to generate murine CD4+CD25+FoxP3+ regulatory T-cells in vivo within OVA sensitized mice. In vivo, pSiNP migrated to the liver, kidneys, lungs, and spleen in both mice and marmosets. Flow cytometry confirmed pSiNP uptake by splenic and peripheral blood DC when functionalized with targeting antibodies. C57BL/6 OVA sensitized mice injected with CD11c-pSiNP loaded with rapamycin + OVA323-339 produced a 5-fold higher number of splenic regulatory T-cells compared to control mice, at 40 days post-pSiNP injection. These results demonstrate the importance of the immobilized targeting antibodies to enhance cellular uptake and enable the in vivo generation of splenic regulatory T-cells.

Characterization of the New Immune Suppressive Anti Human CD83 Monoclonal Antibody 3C12C in Non-Human Primates

Introduction CD83 is a member of the Ig superfamily. It is membrane bound on activated dendritic cells (DC) and B cells. We developed a potential therapeutic human anti-human CD83 monoclonal antibody (3C12C) and showed it prevents graft versus host disease in a human PBMC mouse xenograft model. After establishing that 3C12C binds to non-human primate (NHP, baboon Papio Hamadryas) cells, we tested 3C12C in vivo in NHPs before a first in man, first in class, Phase I clinical trial. Methods Five baboons received 3C12C (1, 5, 10mg/kg, iv) or human IgG 10mg/kg on d0, 7, 14 and d21. Peripheral blood and serum were collected weekly (x4) then every 4 weeks (x2). Bone marrow and lymph node (LN) biopsies were taken at d28. Blood counts and biochemistry were monitored. Flow cytometry analysis followed the PBMC DC subsets, B cells and T cells and bone marrow haematopoietic stem cells. Immune histological studies were performed on LNs. Results All 5 animals remained well following anti-CD83 antibody injection. 3C12C did not change blood counts or liver function. CD4+T, CD8+T and B cells remained normal to d84. 3C12C injection increased peripheral Treg transiently at d21. 3C12C reduced blood CD1c+DC in a dose dependent manner. CD1c+DC were reduced in LN. 3C12C had no influence on bone marrow hematopoietic stem cell numbers. Conclusion Our results demonstrate that 3C12C is safe in NHPs and that it reduced activated DC numbers. This data will facilitate our planned Phase I trial of 3C12C in allogenic hematopoietic stem cell transplantation.

IKKß and Wnt/ß-Catenin Signaling Interaction Regulates Adipogenesis and Osteogenesis in Mesenchymal Stem Cells of Nonhuman Primates and Humans

IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. We recently demonstrated that IKKβ also functions in adipose progenitors to regulate adipogenesis and adipose tissue development in mice. Deficiency of IKKβ in adipose progenitors decreased high-fat (HF)-elicited adipogenesis and protected mice from diet-induced adiposity and insulin resistance. Here we report that IKKβ can regulate both adipogenesis and osteogenesis in mesenchymal stem cells (MSCs) of non-human primates (Macaca fascicularis) and humans. Activation or overexpression of IKKβ increased adipocyte differentiation but inhibited osteoblast differentiation of MSCs isolated from adipose tissue and bone marrow of macaques and humans. By contrast, IKKβ loss of function decreased adipogenesis and increased osteogenesis in macaque and human MSCs. Mechanistically, IKKβ can directly interact with Wnt/β-catenin signaling and phosphorylate β-catenin at serine-33, -37 and -45, leading to its ubiquitination and degradation. Inhibition of canonical Wnt/β-catenin signaling then contributed to the increased adipogenesis and decreased osteogenesis in MSCs. Our findings demonstrate IKKβ as a key molecule that regulates MSC differentiation, and provide a connection between IKKβ and Wnt/β-catenin signaling which may lead to new insights into the pathogenesis of obesity and osteoporosis. Disclosure Y. Sui: None. S. Park: None. P.A. Kern: None. R. Temel: Research Support; Self; Ionis Pharmaceuticals, Inc., Regulus Therapeutics Inc.. Stock/Shareholder; Self; Merck & Co., Inc., Ionis Pharmaceuticals, Inc., Resverlogix Corp.. C. Zhou: None.

Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease.

The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human-but not murine-cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse-but not human-cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain-at least in part-the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses.

Zika Virus Alters the Expression Profile of microRNA-Related Genes in Liver, Lung, and Kidney Cell Lineages.

Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (Flaviviridae). ZIKV infection is associated with alterations in various organs, including the liver, lungs, and kidneys. Studies on the influence of posttranscriptional control on viral infections have demonstrated that microRNAs (miRNAs) interfere with different stages of the replicative cycle of several viruses and may influence the disease outcome. To shed light on ZIKV-induced regulation of host miRNA-processing machinery in the above organs, we analyzed the expression of genes encoding key proteins of the miRNA pathway in different ZIKV-infected continuous primate cell lineages (HepG2, A549, and MA104) by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Expression of the genes encoding the miRNA-related proteins DGCR8, Ago1, and Ago3 in HepG2 cells and Drosha, Dicer, Ago2, and Ago3 in A549 and MA104 cells was significantly altered in the presence of ZIKV. Our results suggest that ZIKV modulates miRNA levels during infection in liver, lung, and kidney cells, which may be an additional mechanism of host cell subversion in these organs.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Methylprednisolone inhibits the proliferation of endogenous neural stem cells in nonhuman primates with spinal cord injury

OBJECTIVE The aim of this work was to investigate the effects of methylprednisolone on the proliferation of endogenous neural stem cells (ENSCs) in nonhuman primates with spinal cord injury (SCI). METHODS A total of 14 healthy cynomolgus monkeys ( Macaca fascicularis) (4-5 years of age) were randomly divided into 3 groups: the control group (n = 6), SCI group (n = 6), and methylprednisolone therapy group (n = 2). Only laminectomy was performed in the control animals at T-10. SCI was induced in monkeys using Allen's weight-drop method (50 mm × 50 g) to injure the posterior portion of the spinal cord at T-10. In the methylprednisolone therapy group, monkeys were intravenously infused with methylprednisolone (30 mg/kg) immediately after SCI. All animals were intravenously infused with 5-bromo-2-deoxyuridine (BrdU) (50 mg/kg/day) for 3 days prior to study end point. The small intestine was dissected for immunohistochemical examination. After 3, 7, and 14 days, the spinal cord segments of the control and SCI groups were dissected to prepare frozen and paraffin sections. The proliferation of ENSCs was evaluated using BrdU and nestin immunofluorescence staining. RESULTS Histological examination showed that a larger number of mucosa epithelial cells in the small intestine of all groups were BrdU positive. Nestin-positive ependymal cells are increased around the central canal after SCI. After 3, 7, and 14 days of SCI, BrdU-positive ependymal cells in the SCI group were significantly increased compared with the control group, and the percentage of BrdU-positive cells in the left/right ventral horns and dorsal horn was significantly higher than that of the control group. Seven days after SCI, the percentages of both BrdU-positive ependymal cells around the central canal and BrdU- and nestin-double positive cells in the left/right ventral horns and dorsal horn were significantly lower in the methylprednisolone therapy group than in the SCI group. CONCLUSIONS While ENSCs proliferate significantly after SCI in nonhuman primates, methylprednisolone can inhibit the proliferation of ependymal cells after SCI.

Interaction of p53 and ASPPs regulates rhesus monkey embryonic stem cells conversion to neural fate concomitant with apoptosis

ABSTRACTThe tumor suppressor p53 is a key regulator of cell apoptosis and cell cycle arrest. Recent studies show that the delicate balance of p53 expression is important for neural tube defects, neuronal degeneration, embryonic lethality, as well as differentiation and dedifferentiation. Moreover, p53 showed different regulatory patterns between rodent and primate embryonic stem cells (ESCs). However, the role of p53 and apoptosis stimulating protein of p53 (ASPP) during neural differentiation (ND) from primate ESCs is still unknown. In this study, using an FGF-2 and/or HGF selectively containing ND culture systems for rhesus monkey ESCs (rESCs), the changes of p53 and ASPPs, and p53 targets, i.e. BAX and p21, were analyzed. Our results showed that the expression patterns of ASPP1/ASPP2 and iASPP were opposite in rESCs but similar in differentiated cells, and the expression of p53 was approximately consistent with BAX, but not p21. These findings indicate that the strong expression of iASPP in ESCs and weak expression of ASPP1/ASPP2 maintain the stability of stemness; and in ND niche, unimpaired iASPP may decrease its inhibition of ASPP1/ASPP2 expression, the interaction of p53 and ASPPs causing rESCs to convert towards a neural fate concomitant with apoptosis, but not to cell cycle arrest.

20th Anniversary of Isolation of Human Embryonic Stem Cells: A Personal Perspective.

Following Jamie Thomson's lecture on primate embryonic stem cells (ESCs) at a meeting I had organized in March 1997, in Israel, to celebrate receipt of the Wolf Prize in Agriculture to my colleague and friend Neal First, frozen human embryos donated for research in Israel were shipped to Wisconsin. The five hESC lines (H1, H7, H9, H13, and H14) were established by early 1998 and transferred to my laboratory just before publication of their existence in Science, on November 6, 1998. The distribution of the cells from my institute to several laboratories, as early as 1999, enhanced the development of hESC research worldwide. My personal perspective regarding the scientific and political events surrounding this story are presented.

Accumulation of follicular CD8+ T cells in pathogenic SIV infection.

LN follicles constitute major reservoir sites for HIV/SIV persistence. Cure strategies could benefit from the characterization of CD8+ T cells able to access and eliminate HIV-infected cells from these areas. In this study, we provide a comprehensive analysis of the phenotype, frequency, localization, and functionality of follicular CD8+ T cells (fCD8+) in SIV-infected nonhuman primates. Although disorganization of follicles was a major factor, significant accumulation of fCD8+ cells during chronic SIV infection was also observed in intact follicles, but only in pathogenic SIV infection. In line with this, tissue inflammatory mediators were strongly associated with the accumulation of fCD8+ cells, pointing to tissue inflammation as a major factor in this process. These fCD8+ cells have cytolytic potential and can be redirected to target and kill HIV-infected cells using bispecific antibodies. Altogether, our data support the use of SIV infection to better understand the dynamics of fCD8+ cells and to develop bispecific antibodies as a strategy for virus eradication.

Ovulatory Induction of SCG2 in Human, Nonhuman Primate, and Rodent Granulosa Cells Stimulates Ovarian Angiogenesis

The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.