Oliver R. Oakley

Periovulatory Leukocyte Infiltration in the Rat Ovary

Ovulation is preceded by intraovarian inflammatory reactions that occur in response to the preovulatory gonadotropin surge. As a main inflammatory event, leukocytes infiltrate the ovary and release proteolytic enzymes that degrade the extracellular matrix weakening the follicular wall, a required step for follicle rupture. This study aimed to quantitatively measure the infiltrating leukocytes, determine their cell types, and localize infiltration sites in the periovulatory rat ovary. Cycling adult and gonadotropin-stimulated immature rats were used as animal models. Ovaries were collected at five different stages of estrous cycle in the adult rats (diestrus, 1700 h; proestrus, 1500 h; proestrus, 2400 h; estrus, 0600 h; and metestrus, 1700 h) and at five different time points after superovulation induction in the immature rats (pregnant mares serum gonadotrophin, 0 h; pregnant mares serum gonadotrophin, 48 h; human chorionic gonadotropin, 6 h; human chorionic gonadotropin, 12 h; and human chorionic gonadotropin, 24 h). The ovaries were either dissociated into a single cell suspension for flow cytometric analysis or fixed for immunohistochemical localization of the leukocytes. Similar numbers of leukocytes were seen throughout the estrous cycle (approximately 500,000/ovary), except proestrus 2400 when 2-fold higher numbers of leukocytes were found (approximately 1.1 million/ovary). A similar trend of periovulatory rise of leukocyte numbers was seen in the superovulation-induced immature rat model, recapitulating a dramatic increase in leukocyte numbers upon gonadotropin stimulation. Both macrophage/granulocytes and lymphocytes were among the infiltrating leukocytes and were localized in the theca and interstitial tissues, where platelet-endothelial cell adhesion molecule-1 and intercellular adhesion molecule-1 may play roles in the transmigration of leukocytes, because their expressions correlates spatiotemporally with the infiltrating leukocytes. In addition, a strong inverse relationship between leukocyte numbers in the ovary and spleen, as well as significant reduction of leukocyte infiltration in the splenectomized rats, were seen, indicating that the spleen may serve as an immediate supplier of leukocytes to the periovulatory ovary.

Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus.

The anti‐inflammatory cytokine interleukin (IL)‐10 plays an important role in the regulation of host‐immune responses. Here we studied the role IL‐10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL‐10 knock‐out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)‐γ, monocyte chemotactic protein 1 (MCP‐1) and IL‐6, all of which are potent stimulators of inflammatory responses. Depletion of IFN‐γ by monoclonal antibodies in IL‐10 knock‐out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL‐6 in the knock‐out animals were unaffected by IFN‐γ depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL‐10 knock‐out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL‐6.

Extra-gonadal sites of estrogen biosynthesis and function

Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496]

Pituitary–ovary–spleen axis in ovulation

Leukocytes are rapidly recruited to the preovulatory ovary and play a crucial role as facilitators of ovulation and luteal formation. In this article, recent findings on leukocyte trafficking to the ovary, as well as the physiological role of leukocytes in the ovary, will be summarized and discussed. We then explore the novel hypothesis that the hypothalamus-pituitary-ovary (HPO) axis might include the spleen as a reservoir of leukocytes by summarizing recent reports on this topic, both in the fields of immunology and reproductive biology.

Inhibition of allogeneic inflammatory responses by the Ribonucleotide Reductase Inhibitors, Didox and Trimidox

BackgroundGraft-versus-host disease is the single most important obstacle facing successful allogeneic stem cell transplantation (SCT). Even with current immunosuppressive therapies, morbidity and mortality rates are high. Current therapies including cyclosporine A (CyA) and related compounds target IL-2 signaling. However, although these compounds offer great benefit, they are also associated with multiple toxicities. Therefore, new compounds with a greater efficacy and reduced toxicity are needed to enable us to overcome this hurdle.MethodsThe allogeneic mixed lymphocyte reaction (MLR) is a unique ex vivo method to study a drugs action on the initial events resulting in T-cell activation and proliferation, synonymous to the initial stages of tissue and organ destruction by T-cell responses in organ rejection and Graft-versus-host disease. Using this approach, we examined the effectiveness of two ribonucleotide reductase inhibitors (RRI), Didox and Trimidox, to inhibit T-cell activation and proliferation.ResultsThe compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-γ, TNF-α, IL-2, IL-13, IL-10 and IL-4.ConclusionsIn conclusion, these data provide critical information to justify further investigation into the potential use of these compounds post allogeneic bone marrow transplantation to alleviate graft-versus-host disease thereby achieving better outcomes.

Increased morbidity and mortality in murine cytomegalovirus-infected mice following allogeneic bone marrow transplant is associated with reduced surface decay accelerating factor expression

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo‐BMT). The manifestations of CMV infection can range from neurological and haematological abnormalities to diminished graft survival and, in extreme cases, death. Many clinical studies have shown a direct correlation between cytomegalovirus infection and increased morbidity and mortality post allo‐BMT, yet the exact mechanism is not well understood. Although driven primarily by T cell responses, the role of complement activation in acute and chronic graft‐versus‐host disease (GVHD) has also become more evident in recent years. The present studies were performed to examine the effects of murine cytomegalovirus (MCMV) infection on decay accelerating factor (DAF) and MCMVs role in exacerbating morbidity and mortality post‐allo‐BMT. Mice infected previously with a sublethal dose of MCMV (1 × 105 plaque‐forming units) have reduced expression of DAF on lung tissues and lymphocytes following allo‐BMT. More importantly, mortality rates post‐allo‐BMT in recipient DAF knock‐out mice receiving wild‐type bone marrow are increased, similar to wild‐type MCMV‐infected recipient mice. Similarly, DAF knock‐out mice showed greater intracellular interferon (IFN)‐γ production by lung CD8 T cells, and infection with MCMV further exacerbated both intracellular IFN‐γ production by CD8 T cells and mortality rates post‐allo‐BMT. Together, these data support the hypothesis that MCMV infection augments morbidity and mortality post‐allo‐BMT by reducing surface DAF expression.

The role of decay accelerating factor in the immunopathogenesis of cytomegalovirus infection.

A wide variety of the host immune elements play an influential role in the defence against cytomegalovirus (CMV) infection. However, the role of complement in the clearance of CMV infection is less well studied. Decay accelerating factor (DAF/CD55) is a membrane‐bound complement regulatory protein that inhibits the formation and accelerates the decay of C3‐convertase. Here we hypothesize that murine CMV (MCMV) utilizes DAF as an immunoevasive strategy through down‐regulation of host adaptive responses against the virus. To test our hypothesis, DAF knock‐out (DAF KO) C57BL/6 mice and wild‐type (WT) littermates were infected with a sublethal dose of MCMV, and their immune responses were compared. WT mice lost 7·8% of their initial weight within the first 4 days after infection and quickly began to recover. This is in contrast to the DAF KO mice, that lost a total of 19·4% of their initial weight and did not start recovery until 6 days post‐infection. Flow cytometric analysis of lung digests revealed that infected DAF KO mice had a significantly increased infiltration of inflammatory cells, the majority being CD8+ T lymphocytes. Serum levels of tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were also increased markedly in the DAF KO mice compared to the infected WT mice. More interestingly, increased viral genome copies (DNA) in the splenocytes of DAF KO mice was accompanied with mRNA transcripts in the DAF KO mice, an indication of active viral replication. These data suggest an intriguing effect of reduced DAF expression on host responses following in vivo MCMV infection.

Paradoxical response to prophylactic Didox (N-3, 4 trihydroxybenzamide) treatment in murine cytomegalovirus-infected mice.

BACKGROUND In this study, we investigated the effect of Didox (DX) on the pathogenicity of and host responses to murine cytomegalovirus (MCMV) infection. METHODS In vitro efficacy of DX against MCMV was determined using plaque reduction assays. For in vivo studies, mice infected with a sublethal dose (10(4) PFU) of MCMV were treated daily with DX (200 mg/kg) using either a prophylactic or delayed protocol. At predetermined intervals, target organs were removed for histopathology. Cytokine transcription and viral load were performed using real-time PCR. Serum cytokine levels were determined by ELISA, and T-cell markers by real-time PCR. RESULTS DX (0.5-50 μM) inhibited MCMV plaque formation in vitro. However, in vivo, prophylactic DX treatment did not decrease viral load and prolonged hepatic proinflammatory cytokine transcription at days 3 and 5 post-infection, which corresponded with more severe histopathological changes observed in the liver. Significant CD8(+) T-cell marker suppression was seen, in accordance with DX-induced inhibition of lymphocyte proliferation observed in vitro. DX prolonged the recovery of MCMV-infected mice when given after infection was established. CONCLUSIONS Despite promising MCMV inhibition in vitro, DX had no beneficial effect on MCMV disease in our model and paradoxically had adverse effects when administered prophylactically. The lack of correlation between in vitro activity and in vivo efficacy emphasizes the importance of selecting appropriate antiviral targets and of using animal models when testing new drugs.