Effectiveness of different treatments for odontogenic keratocyst: a network meta-analysis.

Odontogenic keratocysts (OKC) are benign but aggressive lesions. As there is a lack of well randomized clinical studies assessing the effectiveness of the different treatment options for OKC, a network meta-analysis (NMA) was performed to identify the best treatment option with the lowest recurrence rate. An electronic search was performed following the PRISMA guidelines to identify all clinical studies comparing treatment options against enucleation alone. The outcome variable was recurrence. The predictor variables were treatments. The eight included treatments were: enucleation with peripheral ostectomy/curettage (E + PO/curettage); enucleation with cryotherapy (E + CRYO); enucleation with/without PO followed by modified Carnoy's solution (E ± PO+MCS); enucleation with PO and with topical 5-fluorouracil (E + PO+5FU); enucleation with/without PO followed by original Carnoy's solution (E ± PO+CS); marsupialization alone (MARS); marsupialization followed by secondary enucleation with/without PO (MARS+2°E ± PO); and resection. The odds ratio was used to estimate the recurrence rate. A frequentist NMA was performed using Stata software. A total of 2989 patients in 40 studies were included. Both direct pairwise meta-analysis and NMA showed that E + 5FU+PO was significantly superior to E ± PO+MCS. However, no statistically significant difference was found between E ± PO+CS vs E + 5FU+PO, E ± PO+MCS, and resection, respectively (all very low quality evidence). The three most effective treatments in reducing the recurrence rate were E + PO+ 5FU (98.1%; very low quality evidence), resection (83.5%; very low quality evidence), and E ± PO+CS (63.8%; moderate quality evidence). The findings from this study suggest that CS remains the most effective fixative agent after enucleation and PO until proven otherwise. Additionally, 5FU appears to be an effective method with promising results that needs further research. Finally, the efficacy of MCS remains controversial; further in vivo and in vitro studies are required to determine new protocols. As this NMA included retrospective studies, the results should be interpreted with great caution (level of evidence: type III).

Improving the Physical Health of Psychiatric Hospital Residents: An Evaluation of an Obesity Education Program for Mental Health Professionals.

BACKGROUND: People living with mental health disorders are at increased risk for developing obesity due to poor diet, physical inactivity, and antipsychotic medications. In the United States, the obesity rate is 36% in the general population and more than 50% for people living with mental health disorders. Although mental health clinicians concentrate on managing psychiatric disorders, they seldom recognize the gradual increase in body mass index of their patients. The result is a disconnection between the clinical management of psychiatric disorders and the medical management of obesity. PURPOSE: This study assessed the effectiveness of an evidence-based education program for improving the obesity management practices of mental health clinicians caring for residents at a state psychiatric hospital. METHODS: This was a quasi-experimental study design with a pretest and posttest evaluation. Convenience sampling was used to recruit mental health professionals, or clinicians, at a large psychiatric hospital in the Southern region of the United States. Data was collected with the Advising and Treating Overweight and Obese Patient questionnaire (17 items). Data analysis included descriptive and inferential statistics. The findings were reported in accordance with the TREND and GREET guidelines. RESULTS: The education program was completed by 50 MHCs. The pretest indicated that 76% of MHCs were not involved in helping obese residents manage their weight, but the posttest indicated 90% were involved. There was a significant increase in MHC knowledge about obesity management and reported actions 90-days after the program. MHCs were unable to arrange follow-up visits for residents, a task not directly within their control. CONCLUSIONS: Mental health clinicians reported increased knowledge and improved clinical practice after an education program. Because the outcomes were reported at 90-days after the program, further research needs to evaluate the longitudinal impact of this type of program, where the reported behaviors are correlated to process and clinical outcome measures for obesity.

Assessing Textbook Outcome After Implementation of Transversus Abdominis Release in a Regional Hospital.

Background: The posterior component separation technique with transversus abdominis release (TAR) was introduced in 2012 as an alternative to the classic anterior component separation technique (Ramirez). This study describes outcome and learning curve of TAR, five years after implementation of this new technique in a regional hospital in the Netherlands. Methods: A standardized work up protocol, based on the Plan-Do-Check-Act cycle, was used to implement the TAR. The TAR technique as described by Novitsky was performed. After each 20 procedures, outcome parameters were evaluated and new quality measurements implemented. Primary outcome measure was Textbook Outcome, the rate of patients with an uneventful clinical postoperative course after TAR. Textbook Outcome is defined by a maximum of 7 days hospitalization without any complication (wound or systemic), reoperation or readmittance, within the first 90 postoperative days, and without a recurrence during follow up. The number of patients with a Textbook Outcome compared to the total number of consecutively performed TARs is depicted as the institutional learning curve. Secondary outcome measures were the details and incidences of the surgical site and systemic complications within 90 days, as well as long-term recurrences. Results: From 2016, sixty-nine consecutive patients underwent a TAR. Textbook Outcome was 35% and the institutional learning curve did not flatten after 69 procedures. Systemic complications occurred in 48%, wound complications in 41%, and recurrences in 4%. Separate analyses of three successive cohorts of each 20 TARs demonstrated that both Textbook Outcome (10%, 30% and 55%, respectively) and the rate of surgical site events (45%, 15%, and 10%) significantly (p < 0.05) improved with more experience. Conclusion: Implementation of the open transversus abdominis release demonstrated that outcome was positively correlated to an increasing number of TARs performed. TAR has a long learning curve, only partially determined by the technical aspects of the operation. Implementation of the TAR requires a solid plan. Building, and maintaining, an adequate setting for patients with complex ventral hernias is the real challenge and driving force to improve outcome.

Regulation of bile acid metabolism in biliary atresia: reduction of FGF19 by Kasai portoenterostomy and possible relation to early outcome.

BACKGROUND: Fibroblast growth factor 19 (FGF19) is produced in the small intestine and is involved in suppression of hepatic bile acid (BA) synthesis. FGF19 is also expressed in the liver and serum levels are elevated in adults with cholestatic liver disease. This may reflect a rescue mechanism to dampen liver injury caused by increased intrahepatic BAs. OBJECTIVES: To examine circulating FGF19 at early stages of biliary atresia and at short-term follow-up post-Kasai portoenterostomy (KPE) in relation to noncholestatic infants. The relationship between FGF19, BAs and markers for BA synthesis and hepatic gene expression of factors involved in BA metabolism were also evaluated. METHODS: Liver tissue, portal and peripheral blood samples were obtained from fifteen patients at KPE; additional blood was collected 4-6 months after surgery. Two control groups were included; to examine possible changes related to surgery and to compare FGF19 in biliary atresia to noncholestatic infants. RESULTS: Circulating FGF19 levels correlated to its hepatic gene expression at time of KPE in biliary atresia and levels were elevated compared to noncholestatic infants. At follow-up, FGF19 levels were markedly reduced, and the decline coincided with reductions in bilirubin and conjugated chenodeoxycholic acid and with increased levels of the BA synthesis marker C4. CONCLUSION: Elevated circulating FGF19 in biliary atresia is of hepatic origin and reduced following KPE. Changes in serum FGF19 may reflect the level of restoration of the enterohepatic circulation, and this warrants further long-term studies on the role of FGF19 in the cholestatic liver.

Curcumin induced oxidative stress attenuation by N-acetylcysteine co-treatment: a fibroblast and epithelial cell in-vitro study in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology with only two federally approved drug options. Given the complex molecular pathogenesis of IPF involving multiple cell types and multiple pathways, we explore the effects of a potential antifibrotic and antioxidant drug combination. Curcumin is a polyphenolic compound derived from turmeric with significant biological activity including a potential antifibrotic capacity. N-acetylcysteine (NAC) is a precursor to the antioxidant glutathione. To advance our understanding of these molecules, and to identify a clinical application, we present a small number of focused experiments that interrogates the effect of curcumin and NAC on pathways relevant to IPF in both fibroblasts and epithelial cells. METHODS: Primary epithelial cell and fibroblasts isolated from patients with IPF were challenged with a combination treatment of NAC and curcumin. Evaluation of the antifibrotic potential and effect on oxidative stress was performed through QPCR gene expression analysis and functional assays including scratch tests, viability assays, and measurement of induced reactive oxygen species. RESULTS: We demonstrate that curcumin alone does have antifibrotic potential, but that effect is accompanied by proapoptotic increases in oxidative stress. Coupled with this, we find that NAC alone can reduce oxidative stress, but that epithelial cell viability is decreased through this treatment. However, co-administration of these two molecules decreases oxidative stress and maintains high cell viability in both cell types. In addition, this co-treatment maintains an antifibrotic potential. CONCLUSIONS: These findings suggest a novel application for these molecules in IPF and encourage further exploration of this potential therapeutic approach.

The molecular characterisation of Cryptosporidium species in relinquished dogs in Great Britain: a novel zoonotic risk?

Surveillance was conducted to investigate the occurrence of protozoan parasites of the genus Cryptosporidium in dogs newly admitted to a dog rehoming charity in London, Great Britain. Voided faecal samples were collected from all new admissions between 2011 and 2012 during six separate 4-week sampling periods. Information on host signalment, including age, breed and reason for submission and faecal consistency, was collected. Polymerase Chain Reaction (PCR) targeting the 18S ribosomal RNA gene, confirmed by sequencing, was conducted on the faecal samples to detect Cryptosporidium genomic DNA and determine Cryptosporidium identity. In total, 677 dogs were included in the study. The prevalence of Cryptosporidium-positive faecal samples was 4.6% (31/676). There were positive samples in all of the six sampling periods. Cryptosporidium canis (n = 28), C. parvum (n = 2) and C. andersoni (n = 1) were identified. Sixty KDa glycoprotein (gp60) gene amplicon sequencing of the C. parvum samples identified genotypes IIaA17G1R1 and IIaA15G2R1 for the first time from a dog. There were no significant associations between signalment data and Cryptosporidium status. While this was a study of one rehoming shelter, the presence of the potentially zoonotic C. parvum and C. canis in dogs highlights a public health concern. Further research is needed to better understand the epidemiology and potential impacts of Cryptosporidium infection in dogs.

The impact of nonuniform sampling on stratospheric ozone trends derived from occultation instruments.

This paper applies a recently developed technique for deriving long-term trends in ozone from sparsely sampled data sets to multiple occultation instruments simultaneously without the need for homogenization. The technique can compensate for the nonuniform temporal, spatial, and diurnal sampling of the different instruments and can also be used to account for biases and drifts between instruments. These problems have been noted in recent international assessments as being a primary source of uncertainty that clouds the significance of derived trends. Results show potential "recovery" trends of ∼2-3 % decade-1 in the upper stratosphere at midlatitudes, which are similar to other studies, and also how sampling biases present in these data sets can create differences in derived recovery trends of up to ∼1 % decade-1 if not properly accounted for. Limitations inherent to all techniques (e.g., relative instrument drifts) and their impacts (e.g., trend differences up to ∼2 % decade-1) are also described and a potential path forward towards resolution is presented.

Perforation of the gastric remnant in a patient post-Roux-en-Y gastric bypass.

With the dramatic increase in obesity surgery and the subsequent increase in ageing post-gastric bypass patients, early recognition of possible and serious complications is of the utmost importance. We present a case of a 33-year-old woman who presented to the emergency room, with progressive epigastric pain. The patient had undergone laparoscopic Roux-en-Y gastric bypass surgery 14 months earlier. Diagnostic laparoscopy was performed and showed a prepyloric perforation of the gastric remnant. The defect was closed and omentoplasty was performed. The patient was put on lifelong proton pump inhibitors.

SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma.

Cancer stem cells (CSCs) are a promising target for cancer therapy, particularly for metastatic lung cancers, but how CSCs are regulated is largely unknown. We identify two proteins, SLUG (encoded by SNAI2 gene) and SOX9, which are associated with advanced stage lung cancers and are implicated in the regulation of CSCs. Inhibition of either SLUG or SOX9 sufficiently inhibits CSCs in human lung cancer cells and attenuates experimental lung metastasis in a xenograft mouse model. Correlation between SLUG and SOX9 levels was observed remarkably, we therefore sought to explore their mechanistic relationship and regulation. SLUG, beyond its known function as an epithelial-mesenchymal transition transcription factor, was found to regulate SOX9 by controlling its stability via a post-translational modification process. SLUG interacts directly with SOX9 and prevents it from ubiquitin-mediated proteasomal degradation. SLUG expression and binding are necessary for SOX9 promotion of lung CSCs and metastasis in a mouse model. Together, our findings provide a novel mechanistic insight into the regulation of CSCs via SLUG-SOX9 regulatory axis, which represents a potential novel target for CSC therapy that may overcome cancer chemoresistance and relapse.

De Novo Donor-Specific HLA Antibody Formation in Two Patients With Crigler-Najjar Syndrome Type I Following Human Hepatocyte Transplantation With Partial Hepatectomy Preconditioning.

Clinical hepatocyte transplantation is hampered by low engraftment rates and gradual loss of function resulting in incomplete correction of the underlying disease. Preconditioning with partial hepatectomy improves engraftment in animal studies. Our aim was to study safety and efficacy of partial hepatectomy preconditioning in clinical hepatocyte transplantation. Two patients with Crigler-Najjar syndrome type I underwent liver resection followed by hepatocyte transplantation. A transient increase of hepatocyte growth factor was seen, suggesting that this procedure provides a regenerative stimulus. Serum bilirubin was decreased by 50%, and presence of bilirubin glucuronides in bile confirmed graft function in both cases; however, graft function was lost due to discontinuation of immunosuppressive therapy in one patient. In the other patient, serum bilirubin gradually increased to pretransplant concentrations after ≈600 days. In both cases, loss of graft function was temporally associated with emergence of human leukocyte antigen donor-specific antibodies (DSAs). In conclusion, partial hepatectomy in combination with hepatocyte transplantation was safe and induced a robust release of hepatocyte growth factor, but its efficacy on hepatocyte engraftment needs to be evaluated with additional studies. To our knowledge, this study provides the first description of de novo DSAs after hepatocyte transplantation associated with graft loss.

Stability of bicortical screw versus plate fixation after mandibular setback with the bilateral sagittal split osteotomy: a systematic review and meta-analysis.

The purpose of this study was to test the hypothesis that there is no difference in skeletal stability between bicortical screw and miniplate fixation after mandibular setback surgery with the bilateral sagittal split osteotomy (BSSO). A systematic and electronic search of several databases with specific key words, a reference search, and a manual search through September 2014 was performed. The inclusion criteria encompassed clinical human studies, including randomized controlled trials (RCTs), controlled clinical trials (CCTs), and retrospective studies, with the aim of comparing bicortical screw fixation to miniplate fixation after mandibular setback with the BSSO. Changes in both linear (horizontal and vertical) and angular measurements (SNB and mandibular plane) were analyzed. The initial PubMed search identified 317 studies, of which seven met the inclusion criteria-one RCT, four CCTs, and two retrospective studies. Bicortical screw fixation was found to provide slightly better skeletal stability than miniplate fixation after setback with the BSSO, but the difference was not statistically significant. The results of this meta-analysis support the hypothesis that there is no statistically significant difference in skeletal stability between bicortical screw fixation and plate fixation of the BSSO when used for mandibular setback.

Quetol 651: Not just a low viscosity resin.

Quetol 651, a low viscosity epoxy resin, is miscible with alcohols, acetone, and water. It is versatile and can be used as a single epoxide or mixed with other epoxides and anhydrides. The most important characteristic is that the addition of Quetol 651 to a formulation results in a lower viscosity embedding medium and allows for good detection of antigenic activity. Properly formulated and mixed resins containing Quetol 651 have excellent sectioning properties and good beam stability. The decrease in viscosity lends to lower specific gravity of the embedding medium and less interfering electron density between specimen elements resulting in better spatial resolution. New formulations and viscosity data are presented and compared to long used, embedding formulations and the extensive uses of Quetol 651 are reviewed.

Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

Interferon-gamma release assays piloted as a latent tuberculous infection screening tool in Canadian federal inmates.

BACKGROUND: Interferon-gamma release assays (IGRAs) may be useful in diagnosing latent tuberculous infection (LTBI) in inmates; however, published experience in these settings is limited. OBJECTIVE: To identify variables associated with IGRA positivity among Canadian federal inmates with positive tuberculin skin test (TST) results. DESIGN: On intake, TST-positive (≥10 mm) inmates were offered an IGRA (QuantiFERON(®)-TB Gold), and demographic and historical data were collected. IGRA-positive and -negative inmates were compared using the χ(2) test and multivariable logistic regression; the final model's goodness of fit was assessed using Hosmer-Lemeshow test and area under the receiver operating characteristic curve (AUC). RESULTS: Of 96 TST-positive inmates, 31 (32.3%) were IGRA-positive. Variables associated with positive IGRA were age >45 years (11/20 vs. 20/75, P = 0.016) and previous LTBI treatment (9/20 vs. 13/55, P = 0.032) in univariate analysis, and being from a country with a moderate or high estimated tuberculosis (TB) incidence (OR 3.5, 95%CI 1.3-9.4, P = 0.013) and absence of bacille Calmette-Guérin (BCG) vaccination (OR 3.3, 95%CI 1.2-9.0, P = 0.017) in multivariable analysis. The data fit the model well, classifying the group better than chance alone (AUC 0.67, P = 0.007). CONCLUSION: High discordance with TST, particularly among BCG-vaccinated inmates and those from low TB incidence countries, suggest that IGRA may be useful in Canadian federal penitentiary screening programmes.

Supplemental L-arginine and vitamins E and C preserve xanthine oxidase activity in the lung of broiler chickens grown under hypobaric hypoxia.

The effects of l-Arg, vitamin C (VC), and vitamin E (VE) on xanthine- (XO) and NAD(P)H-oxidase (NOX) activities, and nitric oxide (NO) availability of hypoxic broilers were evaluated. Chickens were kept in wire cages with free access to feed and water. One-day-old chicks were assigned to 1 of 3 diets: control (CTL; ME 3,200 kcal/kg, CP 23%), high Arg (HA; CTL + Arg 0.8%), or high Arg plus VE and VC (AEC; HA + 200 IU of VE/kg of feed + 500 mg of VC/L of water), and grown under hypobaric hypoxia (HYP) from d 7 to 30. A fourth group of birds was fed the CTL diet and grown under normoxia (CTL-NOR). At d 30, chickens were euthanized, their lungs fixed in vivo, excised, and processed for cyto- and histochemistry. The enzymes XO and NOX were localized and activities assessed histochemically and in lung homogenates. The NO depletion was assessed through nitrotyrosine immunocytochemistry colloidal gold particles (NTY). The XO and NOX localized in cell membranes and within vesicles of pulmonary vessel endothelial cells. The XO activity was higher in CTL-NOR birds (586 ± 43 reflectance units) than in both AEC-HYP (456 ± 39) and HA-HYP birds (394 ± 31), whereas CTL-HYP birds had the lowest XO activity (313 ± 27). The NO depletion was not affected by dietary or hypoxia conditions in clinically healthy birds; nevertheless, hypoxic birds that developed pulmonary hypertension had higher NTY levels (less NO, 145 ± 19) than hypoxic but clinically healthy birds (56 ± 11). Thus, the concurrent supplementation of Arg, VE, and VC restored XO activity without affecting NOX activity or NO availability. The dual role of XO, which produces superoxide and uric acid, may have buffered the effects of superoxide in broiler chickens grown under hypobaric hypoxia.

Comparing the detection of iron-based pottery pigment on a carbon-coated sherd by SEM-EDS and by Micro-XRF-SEM.

The same sherd was analyzed using a scanning electron microscope with energy dispersive spectroscopy (SEM-EDS) and a micro X-ray fluorescence tube attached to a scanning electron microscope (Micro-XRF-SEM) to compare the effectiveness of elemental detection of iron-based pigment. To enhance SEM-EDS mapping, the sherd was carbon coated. The carbon coating was not required to produce Micro-XRF-SEM maps but was applied to maintain an unbiased comparison between the systems. The Micro-XRF-SEM analysis was capable of lower limits of detection than that of the SEM-EDS system, and therefore the Micro-XRF-SEM system could produce elemental maps of elements not easily detected by SEM-EDS mapping systems. Because SEM-EDS and Micro-XRF-SEM have been used for imaging and chemical analysis of biological samples, this comparison of the detection systems should be useful to biologists, especially those involved in bone or tooth (hard tissue) analysis.

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

BACKGROUND/OBJECTIVES: Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)2 kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)2 therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)2, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)2 in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate. METHODOLOGY/PRINCIPAL FINDINGS: By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)2 is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)2 binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)2 moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)2 does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes. CONCLUSIONS/SIGNIFICANCE: Together, our results suggest that (KLAKLAK)2 promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)2 moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.

Staining sectioned biological specimens for transmission electron microscopy: conventional and en bloc stains.

Post-staining of ultrathin sections and/or en bloc staining of specimens is necessary for differential contrast and improved resolution of cellular structures. Often specimens are fixed and stained with osmium tetroxide during fixation, but additional contrast is the result of additional heavy metal stains on the sections. The most common post-staining of sections is done on grids by aqueous uranyl acetate followed by lead citrate. When it is apparent that simple, aqueous uranium and lead post-staining is not adequate, other stains are invoked. These procedures can be as simple as en bloc staining with uranyl acetate after primary fixation and osmication. Over the years, several other treatments have been developed for use with the primary fixation or during dehydration. Tannic acid, paraphenylenediamine (PPD), and malachite green can all serve as en bloc stains and can contribute to overall improved visualization of ultrastructural details in biological specimens. Tannic acid and PPD improve membrane preservation, and malachite green is a phospholipid stain. All of these stains are compatible with aqueous fixatives and should be considered when the usual stains are not satisfactory. Marinozzi rings and microwave-assisted post-staining offer alternatives to traditional grid staining. In addition, stain precipitates on grids often can be removed by treatment with 10 % (v/v) acetic acid.

Serum apolipoprotein E as a marker to monitor graft function after hepatocyte transplantation in a clinically relevant mouse model.

INTRODUCTION: Hepatocyte transplantation, a promising treatment for patients with acute hepatic failure or metabolic liver diseases, requires improvement in engraftment as well as long-term function of the liver cells. We established a hepatocyte transplantation model in apolipoprotein E (ApoE) knockout mice, evaluating serum ApoE and lipoprotein profiles as markers of engraftment of transplanted wild-type hepatocytes. Herein we have described a method to monitor the function of transplanted hepatocytes at low levels of engraftment, corresponding to those reported in clinical cases. We also investigated whether pretreatment with anakinra, an anti-interleukin-1 antagonist, methylprednisolone, or a combination of the two agents improved engraftment. METHODS: ApoE (-/-) mice were transplanted with hepatocytes isolated from wild-type C57/bl6 mice. A total of 6 × 10(6) hepatocytes were transplanted by 3 separate intrasplenic injections. Animals were treated before transplantation and daily thereafter for 7 days with anakinra, methylprednisolone, or a combination of both. Graft function was monitored by lipoprotein analysis and quantification of ApoE by enzyme-linked immunosorbent assay. Expression of hepatic ApoE mRNA was quantitated by reverse-transcriptase polymerase chain reaction. RESULTS: Treatment with anakinra with or without methylprednisolone did not significantly increase serum or hepatic mRNA ApoE expression. The low level of hepatocyte engraftment did not normalize lipoprotein profiles, but produced a significant decline in very low-density lipoprotein and total cholesterol. Repeated transplantations significantly enhanced liver repopulation; serum ApoE levels increased with each infusion, correlating well with hepatic mRNA expression. CONCLUSIONS: The model of serum ApoE, a sensitive marker of engraftment and transplanted hepatocyte function, allowed us to study hepatocyte transplantation in a clinically relevant manner, that is, without pretreatments such as retrorsine or carbon tetrachloride.