The effects of hedgehog ligand neutralising antibody 5E1 in a mouse model of endometriosis.

OBJECTIVE: Endometriosis is a common and painful condition characterised by the formation of endometrial lesions within the peritoneal cavity. Previous studies have suggested a role for hedgehog signalling in the pathogenesis of endometriosis. We investigated the role of hedgehog signalling in the establishment of endometriosis lesions using 5E1, a hedgehog ligand neutralising antibody, and a mouse model of endometriosis. To mimic the initiation of endometriosis by retrograde menstruation, which is believed to occur in humans, donor mice underwent an artificial menstruation protocol. Fragments of menstrual endometrium were injected into the peritoneal cavity of estrogen primed recipients. Recipients received twice weekly injections of 5E1 or an isotype matched control antibody for three weeks. Lesions were collected and analysed for markers of epithelium, proliferation and apoptosis by immunofluorescence microscopy. RESULTS: Treatment with 5E1 reduced the number of lesions found on the mesentery. No significant changes were found in the size of lesions, abundance of endometrial epithelial cells, proliferation or apoptosis.

Composite mesh design for delivery of autologous mesenchymal stem cells influences mesh integration, exposure and biocompatibility in an ovine model of pelvic organ prolapse.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.

Recent and emerging reproductive biology research in Australia and New Zealand: highlights from the Society for Reproductive Biology Annual Meeting, 2017.

Research in reproductive science is essential to promote new developments in reproductive health and medicine, agriculture and conservation. The Society for Reproductive Biology (SRB) 2017 conference held in Perth (WA, Australia) provided a valuable update on current research programs in Australia and New Zealand. This conference review delivers a dedicated summary of significant questions, emerging concepts and innovative technologies presented in the symposia. This research demonstrates significant advances in the identification of precursors for a healthy pregnancy, birth and child, and discusses how these factors can influence disease risk. A key theme included preconception parental health and its effect on gametogenesis, embryo and fetal development and placental function. In addition, the perturbation of key developmental checkpoints was shown to contribute to a variety of pathological states that have the capacity to affect health and fertility. Importantly, the symposia discussed in this review emphasised the role of reproductive biology as a conduit for understanding the transmission of non-communicable diseases, such as metabolic disorders and cancers. The research presented at SRB 2017 has revealed key findings that have the prospect to change not only the fertility of the present generation, but also the health and reproductive capacity of future generations.

Heme oxygenase expression in cultured human trophoblast cells during in vitro differentiation: effects of hypoxia.

Heme oxygenases (HO-1 and HO-2) are responsible for the production of carbon monoxide, a vasodilator. HO is important in controlling placental blood flow and expression can be sensitive to oxygen. We previously reported a reduction in HO-2 expression in placentae obtained from patients with pre-eclampsia or living at high altitude, both associated with placental hypoxia. Thus we hypothesized that HO expression in cultured trophoblasts would be altered by exposure to hypoxia. HO-1 and HO-2 expression was assessed in trophoblast cell cultures following exposure to different oxygen environments. Western blot analyses showed that HO-1 expression in syncytiotrophoblast was significantly lower than in cytotrophoblasts in standard conditions (p < 0.05). There was no difference in HO-1 expression in cytotrophoblasts transferred to 2% O2 for various times. However, exposure of syncytiotrophoblast cultures to hypoxia for 12 h resulted in a significant reduction in HO-1 expression (p < 0.05). HO-2 expression was not affected by exposure to hypoxia in either cytotrophoblast or syncytiotrophoblast cultures. Possible interpretations of these findings are that chronic hypoxia alone is not responsible for reduced HO-2 expression or a much longer exposure to chronic hypoxia (perhaps months) is required. This study also reinforces the complexities of HO regulation by oxygen.

Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction.

During early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR.

Treatment of experimental Staphylococcus epidermidis endophthalmitis with oral trovafloxacin.

PURPOSE: To investigate the ocular pharmacokinetics and efficacy of oral trovafloxacin, a novel fluoroquinolone antibiotic, in Staphylococcus epidermidis endophthalmitis. METHODS: Albino rabbits (n = 20) were infected with an intravitreal inoculum of S epidermidis (1.0 x 10(8) colony-forming units [CFU/0.1 ml) and 24 hours later received a single oral dose of trovafloxacin (250 mg/kg). Serum and intraocular samples from infected and control (noninfected) eyes were obtained up to 24 hours after antibiotic administration for measurement of trovafloxacin levels. A second group of rabbits (n = 72) was infected intraocularly and randomized 24 hours later to oral trovafloxacin (250 mg/kg twice a day) for 6 days or no treatment (control). Treatment efficacy was assessed by vitreous culture, clinical examination, and histopathology. RESULTS: Following a single dose of trovafloxacin, maximal vitreous levels were achieved at 8 hours in infected eyes, with a penetration ratio of 36%. Vitreous levels were greater than 15 times the minimum inhibitory concentration of the strain employed. In animals with established endophthalmitis, treated eyes were sterilized after 5 days (P = .0495) compared with control eyes, which autosterilized at 14 days. Clinical and histologic examination revealed significant amelioration of anterior segment inflammation in treated eyes, although severe destruction of posterior segment structures occurred in both groups after 6 days of therapy. CONCLUSIONS: These data support trovafloxacin as a potential oral agent for treatment or prophylaxis of S epidermidis endophthalmitis, although retinal alterations that occur over the period required for vitreous sterilization suggest that it will not replace intravitreal therapy in established endophthalmitis.

Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice.

The transforming growth factor beta 1 (TGF beta 1) signalling pathway is important in embryogenesis and has been implicated in hereditary haemorrhagic telangiectasia (HHT), atherosclerosis, tumorigenesis and immunomodulation. Therefore, identification of factors which modulate TGF beta 1 bioactivity in vivo is important. On a mixed genetic background, approximately 50% Tgfb1-/- conceptuses die midgestation from defective yolk sac vasculogenesis. The other half are developmentally normal but die three weeks postpartum. Intriguingly, the vascular defects of Tgfb1-/- mice share histological similarities to lesions seen in HHT patients. It has been suggested that dichotomy in Tgfb1-/- lethal phenotypes is due to maternal TGF beta 1 rescue of some, but not all, Tgfb1-/- embryos12. Here we show that the Tgfb1-/- phenotype depends on the genetic background of the conceptus. In NIH/Ola, C57BL/6J/Ola and F1 conceptuses, Tgfb1-/- lethality can be categorized into three developmental classes. A major codominant modifier gene of embryo lethality was mapped to proximal mouse chromosome 5, using a genome scan for non-mendelian distribution of alleles in Tgfb1-/- neonatal animals which survive prenatal lethality. This gene accounts for around three quarters of the genetic effect between mouse strains and can, in part, explain the distribution of the three lethal phenotypes. This approach, using neonatal DNA samples, is generally applicable to identification of loci that influence the effect of early embryonic lethal mutations, thus furthering knowledge of genetic interactions that occur during early mammalian development in vivo.

Implication of pneumolysin as a virulence factor in Streptococcus pneumoniae endophthalmitis.

PURPOSE: To determine if pneumolysin, a multifunctional cytotoxin produced by Streptococcus pneumoniae, may be a virulence determinant in the pathogenesis of pneumococcal endophthalmitis. METHODS: Lewis rats (n = 20) were injected intravitreally with purified recombinant pneumolysin at the following doses; 3.9 hemolytic units (HU), 39 HU, 390 HU, 3.9 x 10(3) HU, and 3.9 x 10(4) HU. After 24 hours, eyes were examined clinically and enucleated for histopathologic examination to elucidate the dose-response relationship. To determine the temporal progression of the disease model, a second group of rats (n = 8) were injected intravitreally with 390 HU of pneumolysin. At 6 and 48 hours, eyes were examined clinically and enucleated for histopathology. RESULTS: Eyes injected with pneumolysin demonstrated increasing anterior and posterior segment inflammation in response to increasing doses of administered toxin. The onset of inflammation and tissue damage occurred rapidly, and was maximal at 24 to 48 hours. The clinical and histopathologic changes observed mimicked those of S. pneumoniae endophthalmitis. Histopathologic analysis demonstrated rapid onset of iridocyclitis and vitritis with polymorphonuclear leukocyte influx, inner retinal necrosis, and retinal detachment. Retinal pigment epithelial necrosis and choroiditis were noted at the highest doses administered. Inflamed eyes were shown to be sterile. CONCLUSIONS: Pneumolysin injected intravitreally induces many of the clinical and histopathologic features of pneumococcal endophthalmitis, and may play an important role in the inflammation and tissue damage that occurs in pneumococcal endophthalmitis.

Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice.

Transforming growth factor beta 1 (TGF beta 1) is shown here to be required for yolk sac haematopoiesis and endothelial differentiation. Mice with a targeted mutation in the TGF beta 1 gene were examined to determine the cause of prenatal lethality, which occurs in 50% of homozygous TGF beta 1 null (TGF beta 1-/-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1-+-) conceptions. 50% of TGF beta 1-/- and 25% of TGF beta 1+/- conceptuses were found to die at around 10.5 dpc. The primary defects were restricted to extraembryonic tissues, namely the yolk sac vasculature and haematopoietic system. The embryos per se showed developmental retardation, oedema and necrosis, which were probably secondary to the extraembryonic lesions. The defect in vasculogenesis appeared to affect endothelial differentiation, rather than the initial appearance and outgrowth of endothelial cells. Initial differentiation of yolk sac mesoderm to endothelial cells occurred, but defective differentiation resulted in inadequate capillary tube formation, and weak vessels with reduced cellular adhesiveness. Defective haematopoiesis resulted in a reduced erythroid cell number within the yolk sac. Defective yolk sac vasculogenesis and haematopoiesis were present either together, or in isolation of each other. The phenotypes are consistent with the observation of abundant TGF beta 1 gene expression in both endothelial and haematopoietic precursors. The data indicate that the primary effect of loss of TGF beta 1 function in vivo is not increased haematopoietic or endothelial cell proliferation, which might have been expected by deletion of a negative growth regulator, but defective haematopoiesis and endothelial differentiation.

Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B.

It has previously been suggested that keratinocytes might provide a suitable target cell for delivery of factor IX to the systemic circulation for gene therapy of haemophilia B. Here, an investigation of the use of cellular gene promoters specific for keratinocytes was undertaken to examine whether factor IX could be passed from the epidermis to the systemic circulation. Utilizing two bovine cytokeratin gene promoters, BKIII and BKVI, three lines of transgenic mice were generated with targeted expression of human factor IX in the epidermis. All three transgenic mouse lines secreted epidermally derived human factor IX into the blood system. Most effective factor IX expression (46 ng/ml steady-state levels of circulating human factor IX) was obtained utilizing the BKVI gene promoter, the human homologue of K10, which is expressed exclusively in differentiated keratinocytes, localized distal to the basement membrane. This report demonstrates, for the first time, that human factor IX can be efficiently synthesized and secreted from keratinocytes in situ, and can cross the epidermal basement membrane to reach the systemic circulation. The transgenic mouse model will provide a good in vivo system with which to optimize the efficiency of different keratin gene promoter constructs for delivery of therapeutic gene products to the serum, especially for those promoters, such as K10, which are not effectively expressed in vitro.

Concerted action of TGF-beta 1 and its type II receptor in control of epidermal homeostasis in transgenic mice.

Transforming growth factor-beta 1 (TGF-beta 1) is a modulator of cellular proliferation, differentiation, and extracellular matrix deposition. It is a potent epithelial growth inhibitor and can alter the differentiative properties of keratinocytes, in vitro, but little is known about its normal physiological function in the epidermis in vivo. Transgenic mice were generated using a keratin 10 (K10) gene promoter to drive constitutive expression of TGF-beta 1 in the suprabasal keratinocyte compartment. Surprisingly, these mice showed a two- to threefold increase in epidermal DNA labeling index over control mice, in the absence of hyperplasia. The transgene, however, acted in the expected fashion, as a negative regulator of cell growth, when hyperplasia was induced by treatment by 12-tetradecanoyl-phorbol-13-acetate (TPA). Epidermal TGF-beta type I and II receptor (T beta RI and T beta RII) levels were examined in control and transgenic mice during induction of hyperplasia by TPA. Whereas T beta RI levels remained relatively constant, T beta RII expression was strongly induced in TPA-treated skins, prior to the induction of the growth inhibitory response to TGF-beta 1, and its level of expression correlated with growth sensitivity to TGF-beta 1 in vivo and in vitro. These results suggest that TGF-beta 1 and its type II receptor are part of the endogenous homeostatic regulatory machinery of the epidermis.

Drusen-like deposits in the outer retina of Japanese quail.

Histological examination of the outer retina of Japanese quail (Coturnix coturnix Japonica) ranging in age from 4-48 months revealed the presence of drusen-like deposits which closely resemble those described in primate and human retinas. At both light and electron microscopic levels, these deposits were characterized by randomly distributed, granular and heterogenous materials. Larger deposits with pleomorphic inclusions, often globular in shape, occurred more frequently in older quail, particularly in males. An increase in the incidence and size of drusenoid deposits occurred with increasing age, with a greater rate of accumulation in males than in females. Previously, we have shown that retinal pigment epithelial lipofuscin increases more rapidly with age in female quail and that cumulative light damage is associated with increased lipofuscin in females but not in males. In the present study, no increase in drusen-like deposits was observed in single light-damaged or double light-damaged retinas of either sex. Thus, lipofuscin and the frequency and size of drusenoid deposits do not appear to be directly linked, although both increase with age in both sexes.