Orthopedic Surgery and Anesthesiology Surgical Improvement Strategies Project-Phase II Outcomes.

BACKGROUND: Our institution previously initiated a perioperative surgical home initiative to improve quality and efficiency across the hospital arc of care of primary total knee arthroplasty and total hip arthroplasty patients. Phase II of this project aimed to (1) expand the perioperative surgical home to include revision total hip arthroplasties and total knee arthroplasties, hip preservation procedures, and reconstructions after oncologic resections; (2) expand the project to include the preoperative phase; and (3) further refine the perioperative surgical home goals accomplished in phase I. METHODS: Phase II of the Orthopedic Surgery and Anesthesiology Surgical Improvement Strategies project ran from July 2018 to July 2019. The evaluated arc of care spanned from the preoperative surgical consult visit through 90 days postoperative in the expanded population described above. RESULTS: Mean length of stay decreased from 2.2 days to 2.0 days (P < .001), 90-day readmission decreased from 3.0% to 1.6% (P < .001), and Press-Ganey scores increased from 77.1 to 79.2 (97th percentile). Mean and maximum pain scores and opioid consumption remained unchanged (lowest P = .31). Annual surgical volume increased by 10%. Composite changes in surgical volume and cost reductions equaled $5 million. CONCLUSION: Application of previously successful health systems engineering tools and methods in phase I of Orthopedic Surgery and Anesthesiology Surgical Improvement Strategies enabled additional evolution of an orthopedic perioperative surgical home to encompass more diverse and complex patient populations while increasing system-wide quality, safety, and financial outcomes. Improved process and outcomes metrics reflected increased efficiency across the episode of care without untoward effects. LEVEL OF EVIDENCE: III Therapeutic.

Disseminated protothecosis diagnosed by evaluation of CSF in a dog.

A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.

Pseudorabies virus infection in Oklahoma hunting dogs.

Pseudorabies is caused by Suid herpesvirus 1, a member of the Alphaherpesvirinae subfamily. Although pigs are the natural host of Pseudorabies virus (PRV), the virus has a broad host range and may cause fatal encephalitis in many species. The United States obtained PRV-free status in 2004 after the virus was eradicated from domestic swineherds, but the virus is still present in feral swine populations. The current report describes PRV infection in 3 dogs that were used to hunt feral swine. The dogs developed clinical signs including facial pruritus with facial abrasions, dyspnea, vomiting, diarrhea, ataxia, muscle stiffness, and death. Two were euthanized, and 1 died within approximately 48 hr after onset of clinical signs. The salient histologic changes consisted of neutrophilic trigeminal ganglioneuritis with neuronophagia and equivocal intranuclear inclusion bodies. Pseudorabies virus was isolated from fresh tissues from 2 of the dogs, and immunohistochemistry detected the virus in the third dog. Virus sequencing and phylogeny, based upon available GenBank sequences, revealed that the virus was likely a field strain that was closely related to a cluster of PRV strains previously identified in Illinois. Though eradicated from domestic swine in the United States, PRV is present in populations of feral swine, and should therefore continue to be considered a possible cause of disease in dogs and other domestic animals with compatible clinical history and signs. Continued surveillance is necessary to prevent reintroduction of PRV into domestic swine.

Activation of mutated K-ras in donor endometrial epithelium and stroma promotes lesion growth in an intact immunocompetent murine model of endometriosis.

Endometriosis is a common chronic gynaecological condition, affecting 5-10% of women of child-bearing age. Its study has been hampered by lack of genetically tractable models. We transplanted steroid-manipulated, menstrual-like endometrium from K-ras(G12V/+) /Ah-Cre(+/+) /ROSA26R-LacZ(+/+) mice into gonad-intact immunocompetent wild-type mice. This led to endometriosis-like lesion development. Long-term lesion survival depended on the presence of the activated K-ras in the small proportion of the cells in the mature lesion that had undergone Cre-mediated K-ras activation. LacZ activity demonstrated Cre-mediated recombination in both endometrial epithelial cells and stromal cells, and transgenic K-ras expression was confirmed by RT-PCR. The endometriosis lesions developed without exogenous oestradiol supplementation and anti-oestrogen (fulvestrant, ICI 182780) treatment greatly suppressed their growth. Immunohistochemistry confirmed that as in human endometriosis, there was invasion and activation of fibroblasts, endothelial cells, and macrophages, with marked collagen deposition in the lesions. This model provides an opportunity to investigate endometriosis lesion establishment, growth, and regression in genetically tractable, immunocompetent, and hormonally intact mice. Furthermore, for the first time it provides a suitable model to test clinically validated driver genes in a faithful mouse model of the predisposing endometriotic lesion, thus providing the correct cellular context and microenvironment for ovarian clear cell carcinogenesis.

VEGF-A loss in the haematopoietic and endothelial lineages exacerbates age-induced renal changes.

Renal function declines with age and this is more pronounced in males than females. VEGF-A is essential for glomerular development and function but its role in other aspects of renal function is poorly understood. We therefore investigated the role of VEGF-A, derived specifically from haematopoietic and endothelial lineages in the kidney. We crossed VavCre and floxed Vegf-a mice allowing specific ablation of a single Vegf-a allele in the haematopoietic and endothelial lineages. Mutants were viable and fertile and had normal haematological composition, indicating that 50% gene dosage of Vegf-a in the Vav-expressing lineage is sufficient for establishing a functional haematopoietic system and mature vascular network. However, several abnormalities were observed in the kidney of the adult mutants. These included the formation of inclusion bodies in the proximal tubular cells, tubular atrophy and interstitial fibrosis. These features were observed in 9-11 month-old mutant animals. Most of these abnormalities have been described in aging kidneys in man, and were also observed in the older control mice (24 months). The pathological features appeared in mutant male animals at a younger age than in female mutants. This indicates that reduction in Vegf-a gene dosage in haematopoietic and endothelial lineages accelerates renal aging, suggesting that VEGF-A derived from these lineages may play a role in protecting the kidney from age-associated damage.

Progestin regulates chemokine (C-X-C motif) ligand 14 transcript level in human endometrium.

Leukocyte populations change profoundly in the human endometrium during the menstrual cycle. However the predominant cell, the uterine natural killer (uNK) cell does not contain steroid receptors. From gene array analysis we identified a transcript encoding chemokine (C-X-C motif) ligand 14 (CXCL14) which is markedly up-regulated in the secretory phase of the cycle. We confirm this data by northern blotting and quantitative PCR. Using in situ hybridization we localized CXCL14 mRNA to the glandular epithelial cells where it was detected only in the secretory phase of the cycle. Candidate progesterone response elements were identified at positions -2028/-2007 and -722/-697 (PRE1 and PRE2, respectively) relative to the translation start site. These were functionally tested using luciferase reporter deletion constructs, electrophoretic mobility shift assays and site-directed mutagenesis. The deletion/mutation of these sites reduced progesterone induction by 40 and 20%, respectively. Finally, we demonstrated that recombinant CXCL14 stimulated uNK cell chemotaxis in vitro. We therefore conclude that CXCL14 is likely to be regulated by progesterone in human endometrium and that it may exert a chemoattractive effect on uNK cells and in part be responsible for their clustering around the epithelial glands.

Endometrial-peritoneal interactions during endometriotic lesion establishment.

The pathophysiology of endometriosis remains unclear but involves a complex interaction between ectopic endometrium and host peritoneal tissues. We hypothesized that disruption of this interaction would suppress endometriotic lesion formation. We hoped to delineate the molecular and cellular dialogue between ectopic human endometrium and peritoneal tissues in nude mice as a first step toward testing this hypothesis. Human endometrium was xenografted into nude mice, and the resulting lesions were analyzed using microarrays. A novel technique was developed that unambiguously determined whether RNA transcripts identified via microarray analyses originated from human cells (endometrium) or mouse cells (mesothelium). Four key pathways (ubiquitin/proteasome, inflammation, tissue remodeling/repair, and ras-mediated oncogenesis) were revealed, demonstrating communication between host mesothelial cells and ectopic endometrium. Morphometric analysis of nude mouse lesions confirmed that necrosis, inflammation, healing and repair, and cell proliferation occurred during xenograft development. These processes were entirely consistent with the molecular networks revealed by the microarray data. The transcripts detected in the xenografts overlapped with differentially expressed transcripts in a comparison between paired eutopic and ectopic endometria from human endometriotic patients. For the first time, components of the interaction between ectopic endometrium and peritoneal stromal tissues are revealed. Targeted disruption of this dialogue is likely to inhibit endometriotic tissue formation and may prove to be an effective therapeutic strategy for endometriosis.

Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration.

Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.

Transcript profile and localization of Wnt signaling-related molecules in human endometrium.

The mRNAs encoding several Wnt ligands, Frizzled receptors, and Wnt antagonists were detected in human endometrium, including SFRP1; the levels of SFRP1 mRNA were higher in the proliferative phase of the menstrual cycle and increased in endometriotic tissue compared with control eutopic endometrium, and they were detected in both glandular and stromal cells but were more abundant in the stroma. Our results suggest that Wnt signaling could function in normal endometrial physiology and the the Wnt signalling inhibitor SFRP1 might play a role in endometrial growth and endometriosis development.

Role and regulation of the serum- and glucocorticoid-regulated kinase 1 in fertile and infertile human endometrium.

Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.

Vascular development in embryoid bodies: quantification of transgenic intervention and antiangiogenic treatment.

It has become increasingly clear that the investigation of vascular development is best considered in the context of a whole tissue environment since in vivo endothelial cells interact closely with other cell types. Murine embryoid bodies have been used as a model for the early development of a vascular network and are amenable to genetic manipulation and treatment with soluble modulators. However, quantifying morphological changes in these complex three-dimensional structures is challenging. In this paper we describe protocols to culture embryoid bodies on a large scale to study vascular development together with methods to quantify changes seen when antiangiogenic agents or endothelial cell-specific transgenes are introduced.

Quantitative cellular and molecular analysis of the effect of progesterone withdrawal in a murine model of decidualization.

The endometrium is a dynamic tissue that undergoes periodic growth, remodeling and breakdown under the influence of ovarian steroid hormones. To investigate the molecular mechanisms underlying these processes, we used a murine model to mimic the decidualization and regression observed in humans. Ovariectomized mice were treated sequentially with steroid hormones, and subsequently, to induce decidualization, oil was injected into the uterine lumen. The animals were then divided into progesterone-maintained and progesterone-withdrawal groups. In the latter group, a process similar to menstruation was induced. The uterine tissues were collected at several time-points after the induction of decidualization. Histological analysis demonstrated that decidualization and tissue degeneration were successfully induced with similar features to those observed during the human menstrual cycle. Immunohistochemical, morphometric, and microarray-based techniques were used to study the cellular and molecular changes. The volume fractions of leukocytes, macrophages, and neutrophils, but not endothelial cells, increased in decidualized uteri and decreased after major tissue degradation was completed. The microarray data show that the levels of many transcripts that encode immune-related factors changed during the time-course used for this model, and the transcript levels of many of these factors paralleled the changes observed in the volume fractions of the immune cells. The results of the present study suggest that this model is a useful alternative to the use of non-human primates. Our findings also show that immune cells are recruited into the menstruating endometrium, and that immune-related genes are regulated in the uterus throughout menstruation.

Reduced levels of VEGF-A and MMP-2 and MMP-9 activity and increased TNF-alpha in menstrual endometrium and effluent in women with menorrhagia.

BACKGROUND: Heavy regular menstrual periods (menorrhagia) are an important cause of ill health in women and remain the leading indication for hysterectomy. Abnormalities of the endometrial blood vessels are among the possible causes of this condition. Many different factors affect endothelial cell growth, function and vessel remodelling. We sought to determine whether the levels of vascular endothelial growth factor-A (VEGF-A), tumour necrosis factor-alpha (TNF-alpha), matrix metalloproteinase (MMP)-2 and MMP-9 and soluble VEGF receptor-1 (VEGF-R1) were altered in the menstrual effluent of women with objective menorrhagia. We have also quantitated the VEGF-A mRNA in the menstruated endometrium. METHODS AND RESULTS: We recruited 37 women and determined their menstrual blood loss (MBL) over two cycles and collected menstrual effluent during the 2nd day of bleeding for 4 h. There was no difference in the total level of VEGF-A, and neither latent MMP. However, the concentration of VEGF-A was significantly reduced in the women with menorrhagia, as was the VEGF-A mRNA level. In addition, the active forms of both MMPs were markedly reduced and the total sVEGF-R1 as well as the TNF-alpha content were increased. CONCLUSIONS: This is the first study to show abnormalities of factors important for endothelial cell behaviour in the endometrium of women with menorrhagia. This may underlie the disordered vessel structure and/or function in this condition.

In vitro and in vivo effects of the PPAR-alpha agonists fenofibrate and retinoic acid in endometrial cancer.

UNLABELLED: Fenofibrate, an agonist of PPAR-alpha, in doses above 25 microM, inhibits proliferation and induces apoptosis in Ishikawa endometrial cancer cells. We show that these effects are potentiated by retinoic acid, an agonist of the retinoid-X-receptor. DNA content analysis shows that G1/S phase progression through the cell cycle is inhibited. Independent Component Analysis of gene microarray experiments demonstrated downregulation of Cyclin D1 (CCND1) and associated changes in cell cycle gene expression. Expression of PPAR-alpha mRNA was reduced by >75% using RNA-interference but this resulted in only minor changes in biological effects. A nude mouse model of endometrial carcinoma was used to investigate the effect of fenofibrate in vivo but failed to show consistent inhibition of tumour growth. CONCLUSION: The combination of fenofibrate and retinoic acid is a potent inhibitor of Ishikawa endometrial cancer cell growth in vitro.

Menstrual effluent in endometriosis shows no difference in volume, VEGF-A, MMP2 and MMP9 or sFLT.

Since retrograde menstruation is considered a key event in the aetiology of endometriosis, this study sought to determine whether the menstrual effluent of women with this condition is different from that of those with a normal pelvis. As the amount of blood lost during menstruation is thought to be higher in this group, measured objective menstrual blood loss (MBL) was measured. In addition, factors enhancing both ectopic implantation of endometrium and its subsequent growth (by establishing a neo-vasculature) were chosen for study. Our hypothesis was that they are increased in the menstrual effluent of women with endometriosis. The study showed that at the time of menstruation, there is no difference in MBL or in the volume of menstrual effluent between women with endometriosis and those with a normal pelvis at laparoscopy. In addition, vascular endothelial growth factor-A (VEGF-A) message and protein, soluble truncated receptor sVEGF-R1 (sFLT), matrix metalloproteinase (MMP) 2 and MMP9 activities were also shown to be similar between the two groups. It is concluded that the enhanced expression of VEGF-A and MMP in the peritoneal fluid and ectopic lesions of endometriotic patients may be a secondary event, resulting from an innate difference in peritoneal and systemic factors rather than in the endometrium, causing an abnormal peritoneal response to menstrual debris and facilitating its ectopic implantation.

Identification of genes regulated by interleukin-1beta in human endometrial stromal cells.

Interleukin-1beta (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1beta on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1beta at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1beta. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1beta were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n = 23) and downregulated (n = 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P < 0.02). Our results have identified genes regulated by IL-1beta, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1beta, were verified using real-time PCR. Novel functions regulated by IL-1beta in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1beta in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.

Inhibition of Stat3 activation in the endometrium prevents implantation: a nonsteroidal approach to contraception.

Activation of the receptors for leukemia inhibitory factor (LIF) and IL-11 is essential for embryo attachment and decidualization in mice. Both receptors induce activation of the Stat family of signal transducers via the Jak/Stat pathway. Here, we aimed to establish whether activation of Stat3 in maternal endometrium is essential for successful implantation. Functional blockade of Stat3 before implantation, by injection into the uterine lumen of a cell-permeable Stat3 peptide inhibitor, reduced embryo implantation specifically by 70% (P < 0.001). Stat3 is phosphorylated in the luminal epithelium (LE) in response to LIF, and this phosphorylation was significantly reduced both in vitro and in vivo by the Stat3 inhibitor. The inhibitor also blocked induction by LIF of several LIF-regulated genes in the LE including Irg1, which has been shown previously to be essential for implantation. Successful implantation is therefore dependent on phosphorylation and activation of Stat3 in the endometrium before implantation. This finding provides a target for contraceptive development, based on selective blockade of signal transduction pathways essential for implantation. This study demonstrates that cell-permeable peptide inhibitors can be used effectively to target intracellular signaling pathways in the uterine LE.