Intrapleural tissue plasminogen activator and deoxyribonuclease administered concurrently and once daily for complex parapneumonic pleural effusion and empyema.

BACKGROUND: Pleural infection is life-threatening and increasingly prevalent. In addition to usual care, twice-daily, separate administration of tissue plasminogen activator and deoxyribonuclease (tPA-DNase) reduces radiological pleural opacity with lower surgical referral rates. AIMS: This retrospective cohort study examines the use of once-daily, concurrent administration of tPA-DNase for complex parapneumonic pleural effusion and empyema. METHODS: Patients with pleural infection who received intrapleural tPA-DNase between October 2014 and July 2020 at Logan Hospital, where it is given concurrently and once-daily as salvage therapy, were retrospectively identified. Radiographic opacification, inflammatory markers, clinical response and complications were examined. RESULTS: Thirty-one patients were identified. Mean age was 48.8 years (standard deviation [SD], 17.2). Median tPA-DNase administration was 3 (interquartile range [IQR], 2-3). Chest x-ray pleural opacity decreased significantly (P = 0.047) from a median of 39.6% (IQR, 28.8-65.7%) to 9.7% (IQR, 2.5-23.2%), a median relative reduction of 75.5% (IQR, 47.7-93.9%). White cell count and C-reactive protein improved significantly (P = 0.002 and P = 0.032, respectively) from a median of 16.3 × 109 /L (IQR, 11.8-20.6 × 109 /L) to 9.9 × 109 /L (IQR, 8.0-12.3 × 109 /L) and 311.0 mg/L (IQR, 218.8-374.0 mg/L) to 69.0 mg/L (IQR, 36.0-118.0 mg/L), respectively. No patients experienced significant bleeding or died. Five patients (16.1%) were referred for surgery. CONCLUSION: This is pilot evidence that a practical regimen of concurrent, once-daily intrapleural tPA-DNase improved pleural opacification and inflammatory markers without bleeding or mortality. The surgical referral rate was higher than in studies assessing twice-daily administration, though the validity of this outcome as a measure of treatment success is limited, and further studies are needed to assess the optimal dose and frequency of intrapleural therapy and indications for surgical referral.

Assessing Adult Patients with Facial Deformities for Injectable Treatment: Do Current Classification Systems and Methodologies Meet Important Patient Needs?

BACKGROUND: Many individuals are affected by facial deformities. Injectable aesthetic treatments can often be used to improve appearance and/or dynamic function. However, to best meet the needs of these patients, broadly applicable methodologies are required for classifying the deformity, assessing severity, and developing a treatment strategy. OBJECTIVE: To assess whether any published systems could be used for this purpose. METHODS: Thirty-eight searches were conducted in PubMed (1999-2019; in English). Forty-two publications were identified describing novel classification systems for adult facial deformity. They were analyzed against a checklist of 10 characteristics defining an "optimal" system-based on appropriate anatomical coverage, wide usability across types of deformity, user-friendliness, applicable underlying methodology, and ability to guide treatment with injectables. RESULTS: None of the systems met more than 7 of the 10 checklist criteria; none were usable across multiple types of deformity or provided a recommendation for treatment with injectables. CONCLUSION: There remains a need for a broadly applicable system for classifying adult facial deformities ahead of injectable therapy. The checklist provides a developmental framework. With the increasing popularity and accessibility of injectables, this diverse and complex demographic is at risk of mismanagement without superior methods for devising treatment strategies.

Treatment of Horizontal Wrinkles of the Neck Using a Hyaluronic Acid Filler: Results From a Prospective Study.

BACKGROUND: VYC-12 is a hyaluronic acid filler with low cohesivity. OBJECTIVE: To evaluate the safety and effectiveness of VYC-12 for aesthetic improvement of horizontal neck lines. METHODS: This was a prospective study of consecutive women undergoing neck treatment using VYC-12. All had a baseline score of 1 to 4 on the Allergan Transverse Neck Lines Scale (ATNLS). Individuals with an ATNLS score of 1 to 3 were treated with VYC-12 alone; those with a score of 4 received filler combined with high-intensity focused ultrasound (HIFU). Total VYC-12 volumes were ∼1 mL per patient. Follow-up lasted ≤30 months. RESULTS: Fifty women were enrolled (mean age: 55.0 ± 5.7 years; n = 42 VYC-12 alone, n = 8 VYC-12 + HIFU). Forty-six patients (92%) achieved a ≥ 1-grade improvement on ATNLS 1 month post-treatment; the mean ATNLS score decreased from 2.64 ± 0.83 to 1.44 ± 0.81 (p < .0001). Rasch-transformed scores on the FACE-Q "Appraisal of the Neck" questionnaire improved from 31.0 ± 14.2 at baseline to 49.7 ± 14.4 at 1 month (p < .0001). Repeat injections at 9- to 12-month intervals led to progressive improvements. There were no treatment-related adverse events. CONCLUSION: Treatment of the neck using VYC-12 was safe and effective in reducing the appearance of horizontal lines.

Skin Quality Improvement With VYC-12, a New Injectable Hyaluronic Acid: Objective Results Using Digital Analysis.

BACKGROUND: VYC-12 is a novel hyaluronic acid-based dermal filler designed to treat fine lines and improve skin quality. A specialist digital camera and proprietary Digital Analysis of the Cutaneous Surface (DACS) software have previously been used to objectively measure changes in skin features. OBJECTIVE: To assess the effect of facial treatment with VYC-12 on skin texture using the specialist camera. MATERIALS AND METHODS: This was a prospective, open-label, 2-center study of 40 women aged 35 to 60 years treated with multiple, microdepot intradermal injections of VYC-12 (2 mL in the face; 1 mL in the neck if required). Eight patients (20.0%) required a touch-up at Day 45. Images were acquired using the specialist camera at baseline and 45 days and 6 months after treatment, and were analyzed by DACS. Clinical improvements were also assessed subjectively using the Global Aesthetic Improvement Scale (GAIS). RESULTS: VYC-12 improved skin texture from baseline after 45 days (mean improvement: 25.9% ± 9.2%) and 6 months (mean improvement: 30.7% ± 18.2%). Improvements were also evident using the GAIS. There were no major adverse events. CONCLUSIONS: VYC-12 improves skin quality, as measured using an objective, fast, and reproducible measuring tool. VYC-12 represents a valuable addition to the treatment armamentarium.

Impaired steroidogenesis and apoptosis of granulosa-luteal cells in primary culture induced by cis-platinum.

OBJECTIVE: The purpose of this study was to test the hypothesis that the cytotoxic drug cis-platinum (CP) induces premature ovarian failure by reducing the viability of human granulosa cells. STUDY DESIGN: We incubated cultured human granulosa-luteal cells (GLCs) with varying concentrations of CP for 48 hours. Steroidogenesis and apoptosis were assessed by progesterone and estradiol, annexin V/propidium iodide, phase contrast, and transmission electron microscopy. RESULTS: CP caused impaired production of progesterone and estradiol in a dose- and a time-dependent fashion. The estradiol production was more pronounced than progesterone for each concentration of CP that was studied. The phase contrast microscopy of CP-treated GLCs showed loss of cell number with condensed nuclei. CP-induced apoptosis was maximum at 20 µg/mL compared with a 10-µg/mL concentration (79.9% ± 4.6% vs 58.3% ± 3.9%; P < .01). The hallmark of apoptosis (ie, nuclear condensation, cell size shrinkage) was seen in CP-treated cells by transmission electron microscopy. CONCLUSION: CP induces apoptosis of human GLCs in culture with impaired steroidogenesis, which may be one mechanism by which a CP-containing regime induces premature ovarian failure.

Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.

In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

Comparison of NMR lipid profiles in mitotic arrest and apoptosis as indicators of paclitaxel resistance in cervical cell lines.

This study aimed to characterize changes in lipid saturation using magnetic resonance spectroscopy of sensitive (HeLa) and resistant (C33A; Me180) cervical cancer cell lines following exposure to paclitaxel to explore lipid profiles as biomarkers of drug resistance. Spectra were acquired at 11.74 T. Flow cytometry, electron, and confocal microscopy assessed cellular morphology. Western blots assessed cytoplasmic phospholipase A(2) , fatty acid synthase, and acyl-CoA synthetase1 expression. After 24 h of paclitaxel exposure, >60% of cells showed mitotic arrest. At 48 h, HeLa cells showed apoptosis while C33A/Me180 cells showed normal morphology indicating resistance. MR-visible lipids increased significantly in all lines at 24 h with further increases at 48 h; resistant lines showed smaller increases than HeLa. Cytoplasmic phospholipase A(2) and fatty acid synthase levels were unchanged at 24 h and dropped at 48 h in HeLa; acyl-CoA synthetase1 was higher in Me180/C33A than in HeLa controls but did not increase significantly. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines; droplet size increased only in HeLa cells. Droplet number was >3-4× greater in apoptotic compared with mitotic-arrested cells. Apoptotic cells accumulate unsaturated fatty acids in large (relative to control) droplets; resistant lines accumulated smaller droplets with less triglycerides.

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice.

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.

Investigation of tumor-peritoneal interactions in the pathogenesis of peritoneal metastases using a novel ex vivo peritoneal model.

BACKGROUND: Peritoneal metastasis occurs in up to 30% of patients with gastric cancer. The aim of this experimental study is to develop and validate a novel ex vivo model of the human peritoneum to better identify factors involved in the development of peritoneal metastasis in order to improve its management and prognosis. METHODS: Peritoneal discs harvested from hernia sacs obtained at inguinal hernia surgery were suspended in media using Teflon rings. Viability of the tissue was investigated using MTS assay, light and scanning electron microscopy (LM and SEM) over 72 h. To assess validity of the model, phenotypic changes in tumor cells were investigated. Changes in matrix metalloproteinases (MMP)-2 and -9 activities in HGC and AGS gastric adenocarcinoma cells after co-culture were investigated using zymography. Modulation of tumor cell adhesion to peritoneum after exposure to heparin was assessed using a fluorometric adhesion assay. Analysis was performed using Kruskal-Wallis for multiple comparisons and Mann-Witney U for comparisons between each group. RESULTS: MTS assay showed reduced viability after 72 h (P = 0.047, compared with 24 h). Mesothelial cell loss at 48 h was demonstrated by LM and SEM, confirming peritoneal viability for at least 24 h after tissue harvesting. Zymography confirmed increased MMP2 and -9 activities in tumor cells and peritoneal tissue during co-culture compared with controls, and heparin significantly reduced tumor cell adherence (P = 0.04), as observed in published in vivo models. CONCLUSION: A validated complete model of peritoneum was developed that has shown potential to determine realistic mechanisms of peritoneal metastasis.

Elevated expression of the metabolic regulator receptor-interacting protein 140 results in cardiac hypertrophy and impaired cardiac function.

AIMS: Receptor-interacting protein 140 (RIP140) is a ligand-dependent cofactor for nuclear receptors that regulate networks of genes involved in cellular processes, including metabolism. An important role for RIP140 in metabolic control has been identified in RIP140 null mice, whose phenotypes include derepression of genes involved in energy mobilization or catabolism in adipocytes and a switch to more oxidative fibres in skeletal muscle. We hypothesized that ubiquitous expression of RIP140 would suppress metabolic processes, leading to defects in development or cellular function. METHODS AND RESULTS: The primary effect of exogenous expression of RIP140 mRNA (real-time PCR) and protein (western blotting) in transgenic mice is impaired postnatal heart function. There was rapid onset of cardiac hypertrophy and ventricular fibrosis, detected microscopically, in male RIP140 transgenic mice from 4 weeks of age, resulting in 25% mortality by 5 months. RIP140 exogenous expression in the heart leads to decreased mitochondria state III and state IV membrane potential and oxygen consumption. Quantitative PCR showed more than 50% reduced expression of genes involved in mitochondrial activity and fatty acid metabolism, including mitochondrial transcription factor A, cytochrome oxidase VIIa, cytochrome XII, CD36, medium-chain acyl dehydrogenase, and fatty acid transport protein, many of which are known targets for nuclear receptors, including peroxisome proliferator-activated receptors PPARalpha and PPARdelta and oestrogen-related receptors ERRalpha and ERRgamma. CONCLUSION: This study demonstrates that RIP140 is an important cofactor in postnatal cardiac function and that inhibition of the action of RIP140 may provide a model system to investigate specific interventions designed to prevent or delay the onset of cardiac disease.

Progesterone induces nano-scale molecular modifications on endometrial epithelial cell surfaces.

BACKGROUND INFORMATION: The endometrial epithelial cell membrane is a key interface in female reproductive biology. Steroid hormones play a predominant role in cyclic changes which occur at this interface during the female menstrual cycle. Specific changes in the morphology of the endometrial epithelial cell surface become apparent with the epithelial transition that drives the switch from a non-receptive to receptive surface due to the action of progesterone on an oestrogen primed tissue. AFM (atomic force microscopy) allows the high-resolution characterization of the endometrial epithelial cell surface. Its contact probe mechanism enables a unique imaging method that requires little sample preparation, yielding topographical and morphological characterization. By stiffening the cell membrane, low concentrations of fixatives allow the surface detail of the cell to be resolved while preserving fine ultra-structural details for analysis. RESULTS: In the present study we use high resolution AFM analysis of endometrial epithelial cells to monitor the effect of progesterone on the nanoscale structure of the endometrial cell surface. High-resolution imaging reveals similar topographical nanoscale changes in both the Hec-1-A and Ishikawa model cell lines. Hec-1-B cells, used in the present study as a progesterone receptor negative control, however, exhibit a flattened cell surface morphology following progesterone treatment. Changes in average cell height and surface convolution correlate with increased surface roughness measurements, demonstrating alterations in molecular structure on the cell surface due to hormonal stimulation. CONCLUSIONS: Progesterone treatment induces changes to the cell surface as a result of nanoscale molecular modifications in response to external hormonal treatments. AFM provides the basis for the identification, visualization and quantification of these cell surface nanoscale changes. Together these findings demonstrate the utility of AFM for use in reproductive science and cancer biology where it could be applied in both in vitro analysis of protein structure-function relationships and clinical diagnosis.

Effect of cell shape and packing density on granulosa cell proliferation and formation of multiple layers during early follicle development in the ovary.

The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.

The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle.

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

Analysis of epitopes on endometrial epithelium by scanning immunoelectron microscopy.

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and "ciliogenic" cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.