In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in ...parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1β) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1β significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1β stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo.
This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1β did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.
The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). ...We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.
Hibernoma is an uncommon benign fatty tumor that arises from the vestiges of fetal brown fat. We present a case report of a hibernoma of the back in a symptomatic 42-year-old man and describe the ...important clinical, histopathologic, and imaging findings. Computed tomography shows a well-defined hypodense mass with septations. Magnetic resonance imaging shows intermediate T1 and bright T2 signal of the mass and also demonstrates the characteristic marked contrast enhancement.
To determine if disturbances of the liver microcirculation may be of pathophysiological relevance for liver damage during acute biliary obstruction, we studied the effects of bile duct ligation (BDL) ...on hepatic microhemodynamics and leukocyte adhesion in rat liver in vivo. Male Wistar rats were subjected to BDL for 3 days and 7 days, respectively. Sham‐operated controls underwent laparotomy without BDL. After 3 days, intravital fluorescence microscopy (IVM) and hydrogen gas (H2) clearance were performed to study hepatic microvascular perfusion. Furthermore, leukocyte‐endothelial cell interactions were assessed by IVM. Intercellular adhesion molecule 1 (ICAM‐1) protein expression was studied by Western blot analysis and tissue immunofluorescence after 3 and 7 days, respectively. Analysis of microvascular perfusion by IVM revealed a marked impairment of sinusoidal perfusion after 3 days. Assessment of H2 clearance confirmed that overall hepatic microvascular perfusion was decreased. In addition, increased leukocyte adhesion in sinusoids and venules could be observed. A concomitant increase of ICAM‐1 expression in liver tissue was also noted within the first week after BDL. Our results show that BDL is followed by a marked depression of the hepatic microcirculation and increased leukocyte adhesion in vivo within 3 to 7 days. Together, these findings suggest that deficits in microvascular perfusion and increased neutrophil infiltration may represent a potential source of liver injury during acute biliary obstruction.
Objectives/Hypothesis
To determine whether there is an association between the geographic location of an applicant's undergraduate school, medical school, and residency program among matched ...otolaryngology residency applicants.
Study Design
Observational.
Methods
Otolaryngology residency program applications to our institution from 2009 to 2013 were analyzed. The geographic location of each applicant's undergraduate education and medical education were collected. Online public records were queried to determine the residency program location of matched applicants. Applicants who did not match or who attended medical school outside the United States were excluded. Metro area, state, and region were determined according to US Census Bureau definitions.
Results
From 2009 to 2013, 1,089 (78%) of 1,405 applicants who matched into otolaryngology residency applied to our institution. The number of subjects who attended medical school and residency in the same geographic region was 241 (22%) for metropolitan area, 305 (28%) for state, and 436 (40%) for region. There was no difference in geographic location retention by gender or couples match status of the subject. United States Medical Licensing Exam step 1 scores correlated with an increased likelihood of subjects staying within the same geographic region (P = .03).
Conclusions
Most otolaryngology applicants leave their previous geographic area to attend residency. Based on these data, the authors recommend against giving weight to geography as a factor when inviting applicants to interview.
Level of Evidence
NA Laryngoscope, 126:829–833, 2016
Anabolic activity, phenotypic alterations, and in particular survival of the chondrocytes are essential for the maintenance of proper articular cartilage and appears to fail during osteoarthritic ...cartilage degeneration. In this study, we investigated the presence and expression of RhoB in adult human articular cartilage and its regulation in osteoarthritic cartilage as well as in chondrocytes in vitro. RhoB belongs to the family of small GTPases, which are thought to be involved in a large range of activities important for eukaryotic cells. Conventional and quantificative PCR analysis showed significant levels of RhoB expression in normal articular cartilage. Immunolocalization and confocal laser scanning microscopy showed strong cytoplasmic signals for RhoB in normal chondrocytes. In osteoarthritic cartilage, a significantly lower expression of RhoB was detectable. In vitro experiments showed a quick (and transient) up-regulation of RhoB after stimulation with interleukin-1beta and serum. Our study suggests that RhoB is constitutively expressed and essential for adult articular chondrocytes, but significantly down-regulated in osteoarthritic chondrocytes. One intriguing speculation might be that the down-regulation of RhoB in osteoarthritic chondrocytes is at least partly a prerequisite for the sustained pre- or para-apoptotic phenotype of osteoarthritic chondrocytes, because RhoB is known to be one important molecule in the induction of apoptotic cell death in response to DNA damage and osteoarthritic chondrocytes are known to have significant DNA damage. Alternatively, RhoB could be involved in the activation or deactivation and the destabilization of the functional phenotype of chondrocytes in osteoarthritic joint degeneration Thirdly, RhoB is associated with the cell cycle, which is re-initiated in osteoarthritis.
Orthotopic liver transplantation (OLT) in rat is a demanding procedure, which has become a popular model to investigate various problems. Our aim was to review and analyze the various techniques of ...experimental OLT in the rat. A review of the literature revealed 30 techniques or technical modifications. Each modification represented a change or a simplification of the reconstruction method of five anatomical structures, which are cornerstones of a successful OLT: the suprahepatic inferior vena cava (SHVC), portal vein (PV), infrahepatic inferior vena cava (IHVC), hepatic artery (HA), and bile duct (BD). SHVC is anastomosed via microsuture or cuff. The PV anastomosis is performed by microsuture, cuff, or a microsuture-temporary splint technique. IHVC is reconstructed by a microsuture, cuff, or microsuture-temporary splint technique. Arterialization has been accomplished via microsuture (aortic segment, celiac segment, or aortic patch), cuff, splint, sleeve, or telescopic method. Nonarterialization of the graft has also been described. Methods for BD reconstruction include pull-through, telescopic, splint, and T-tube. Although a high level of microsurgical skill is the basic requirement in the microsuture technique which provides the most physiological situation and concomitantly reduces thrombosis, it increases anhepatic time compared to the cuff procedure. The learning curve of microsuture techniques is flat; beginners need much practice to become expert. The most physiologic techniques for anastomoses are preferred for long-term survival studies, while the faster techniques are options for short-term survival studies. Each research group must choose techniques according to study defined aims.
Protective effects of desmopressin in brain dead organ donors oppose reports on a hypercoagulatory potential and an increased leukocyte-endothelial interaction (LEI) after application of the drug. ...The aim was to evaluate the effect of desmopressin on organ donor's pancreas and early graft function.
Donor microcirculation was evaluated via intra-vital microscopy (IVM) in 24 BR (di/di) rats with central diabetes insipidus, randomly assigned to groups I (control without desmopressin application), II (single i.v. application, no pretreatment) or group III (single i.v. desmopressin application, s.c. pretreatment for 3 days). Microcirculation in recipients was evaluated 1 hr and 6 hr after syngenic pancreas transplantation. Groups III and I served as organ donors. After IVM specimens were taken for histology and immunohistochemistry.
Desmopressin in II vs. I led to temporarily (30') increased LEI (Sticker 274.3+/-87.7 vs. 76.5+/-31.1/mm2 endothelial surface; P<0.01) and impaired microcirculation (MCEV 0.43+/-0.07 vs. 0.99+/-0.06 mm/s; P<0.01). Repeated application reduced MCEV and increased LEI for up to 12 hr. Histology in I vs. III showed increased inflammation (n.s.), necrosis (P<0.05) and vacuolization (P<0.01). Immunohistochemistry revealed increased endothelial P-selectin 20' after application. 6 hr after reperfusion organs from III showed reduced MCEV and increased LEI (P<0.01).
Repeated application of desmopressin impairs graft microcirculation. Perfusion of the pancreas is significantly reduced at the beginning of organ tissue conservation as well as after reperfusion. These disturbances might partly be due to observed endothelial P-selectin expression. Application of desmopressin up to 12 hr prior to organ explantation may impact graft quality.
Recent studies provide evidence that nitric oxide (NO) has beneficial effects in hepatic ischemia/reperfusion injury. The purpose of this study was to evaluate whether nitric oxide is involved in the ...regulation of hepatic microvascular perfusion after warm hepatic ischemia. Therefore, we performed a study using in vivo fluorescence microscopy.
Clamping of the left liver lobe was performed in male Wistar rats for the duration of 70 min. One experimental group (n=8) received L-NAME (Nw-nitro-L-arginine methyl ester hydrochloride), an NO-synthase inhibitor, 1 min prior to reperfusion. A second experimental group (n=8) received L-arginine (NO-substrate) continuously infused throughout the observation period. Controls (n=8) received equivalent volumes of an isotonic solution and underwent the same procedures. Hepatic microvascular blood flow and leukocyte-endothelial cell interaction was studied between 20 and 90 min after reperfusion using in vivo fluorescence microscopy.
Inhibition of NO-synthesis during reperfusion by application of L-NAME caused a marked decrease in sinusoidal blood flow velocity. Furthermore, we noted an increase of non-perfused sinusoids in this group. Treatment with L-arginine improved functional perfusion of hepatic acini and reduced significantly the number of adherent leukocytes in sinusoids and venules compared to control animals.
Our results provide further evidence that NO maintains postischemic hepatic microvascular perfusion and that inhibition of NO synthesis has detrimental effects on hepatic microhemodynamics during reperfusion.
Electrical cell uncoupling via gap junction closure is assumed to cause characteristic changes of the passive dielectric spectrum of ischemic heart tissue. In order to find an independent evidence ...for this assumption, we analysed heart tissue during ischemia, measured the open state of gap junctions by means of dye transfer and correlated this parameter with the time course of the dielectric permittivity.
The hearts were preischemically arrested by perfusion with Ringer solution containing 20 mmol/L of potassium (group KCL,
n=10). This solution was also used with the addition of two gap junction blockers, either 3 mmol/L heptanol (group HEP,
n=4) or 20 μmol/L palmitoleic acid (group PA,
n=7). During subsequent ischemia at 21.0±0.5 °C, we monitored the passive dielectric permittivity spectrum and the spread of dye.
After a sigmoidal increase the dielectric permittivity reached an upper plateau at 61±22 min of ischemia in KCL, at 45±7 min in PA, and already during perfusion at 2±1 min in group HEP. At the beginning of ischemia, dye migrated to neighbouring cells in groups KCL and PA but not in HEP. In KCL and PA, the intercellular diffusion of dye stopped after 64±26 and 40±11 min of ischemia, respectively.
Our results suggest that the sigmoidal increase in dielectric permittivity and the reduction of dye diffusion depend on a common mechanism, namely gap junction closure.
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