Structure-dependent behaviours of skin layers studied by atomic force microscopy.

The multilayer skin provides the physical resistance and strength against the environmental attacks, and consequently plays a significant role in maintaining the mammalian health. Currently, optical microscopy (OM) is the most common method for the research related to skin tissues while with the drawbacks including the possibility of changing the native morphology of the sample with the addition of the chemical or immunological staining and the restricted resolution of images for the direct observation of the tissue structures. To investigate if the function of each tissue is structure-dependent and the how the injured skin returns to the intact condition, we applied atomic force microscopy (AFM) on the sectioned mice-skin to reveal the tissue structures with a nanoscale resolution. From the outermost stratum to the inner layer of the skin tissue, the respectively laminated, fibrous, and brick-like structures were observed and corresponded to various functions. Due to the mechanical differences between the tissue constituents and their boundaries, the sizes and arrangements of the components were characterised and quantified by the mechanical mapping of AFM, which enabled the analytical comparisons between tissue layers. For the wound model, the skin tissues were examined with the initial formation of blood vessels and type-I collagen, which agreed with the stage of healing process estimated by OM but showed more detail information about the evolution of proteins among the skin. In conclusion, the characterisation of the components that consist of skin tissue by AFM enables the connection of the tissue function to the corresponded ultrastructure.

Non-operative management of fingertip injuries with an intravenous dressing.

OBJECTIVE: The optimal management of fingertip injuries is a much debated topic. Surgical and nonsurgical options, including treatment with dressings alone, have comparable results. IV3000 is a semi-occlusive dressing with a high reactive moisture vapour transmission rate (MVTR) compared to its alternatives. As the fingertip is crucial to hand function, determining the optimal dressing to treat these injuries is of clinical importance. The aim of this study is to collect preliminary data on the IV dressing when used to treat fingertip injuries. METHOD: Patients were recruited from the department of orthopaedic surgery outpatient clinic. Inclusion criteria were a fingertip injury with skin loss and emergency department treatment consistent with the study protocol, including washing the fingertip, simple debridement as required, administration of antibiotics, tetanus prophylaxis, and fingertip dressed with the IV dressing. RESULTS: Fingertip injuries (15) from 13 male patients were identified. With the exception of one, all injuries were treated with the IV dressing and were included in the analysis. The treatment outcome of 13 injuries was rated as 'satisfactory' by the patients, while one was rated 'indifferent'. The latter was on one of two patients with injuries to two digits. No patient reported their outcome as 'unsatisfactory'. At the 18-24 months' follow-up, seven of the 14 affected digits had some degree of hypersensitivity, eight regained normal pulp thicknesses, one had thickened padding, and five had reduced pulp volume. All but one patient reported some degree of numbness. Nail involvement was seen in 11 injuries, all of which continued to have some degree of nail deformity. CONCLUSION: The IV dressing provides satisfactory outcomes when used to treat fingertip injuries. As the dressing possesses properties that suggest it would result in a superior healing environment compared to other semi-occlusive dressings, a prospective, randomised control trial should be conducted to determine whether these properties translate into superior outcomes when used to treat fingertip injuries.

Decreased core muscle size is associated with worse patient survival following esophagectomy for cancer.

Preoperative risk assessment, particularly for patient frailty, remains largely subjective. This study evaluated the relationship between core muscle size and patient outcomes following esophagectomy for malignancy. Using preoperative computed tomography scans in 230 subjects who had undergone transhiatal esophagectomy for cancer between 2001 and 2010, lean psoas area (LPA), measured at the fourth lumbar vertebra, was determined. Cox proportional hazards regression was employed to analyze overall survival (OS) and disease-free survival (DFS) adjusted for age, gender, and stage, and the Akaike information criterion was used to determine each covariate contribution to OS and DFS. Univariate analysis demonstrated that increasing LPA correlated with both OS (P = 0.017) and DFS (P = 0.038). In multivariate analysis controlling for patient and tumor characteristics, LPA correlated with OS and DFS in patients who had not received neoadjuvant treatment (n = 64), with higher LPA associated with improved OS and DFS. Moreover, LPA was of equivalent, or slightly higher importance than pathologic stage. These measures were not predictive among patients (n = 166) receiving neoadjuvant chemoradiation. Core muscle size appears to be an independent predictor of both OS and DFS, as significant as tumor stage, in patients following transhiatal esophagectomy. Changes in muscle mass related to preoperative treatment may confound this effect. Assessment of core muscle size may provide an additional objective measure for risk stratification prior to undergoing esophagectomy.

Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development.

Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1-dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes.

Respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study.

OBJECTIVES: To determine the viral epidemiology and clinical characteristics of patients with and without clinically apparent respiratory tract infection. METHODS: This prospective cohort study was conducted during the 2018 winter influenza season. Adult patients with fever/respiratory symptoms (fever/RS group) were age- and sex-matched with patients without fever/RS (non-fever/RS group) in a 1:1 ratio. Respiratory viruses were tested using NxTAG™ Respiratory Pathogen Panel IVD, a commercially-available multiplex PCR panel. RESULTS: A total of 214 acutely hospitalized patients were included in the final analysis, consisting of 107 with fever/RS (fever/RS group), and 107 age- and sex-matched patients without fever/RS (non-fever/RS group). Respiratory viruses were detected in 34.1% (73/214) of patients, and co-infection occurred in 7.9% (17/214) of patients. The incidence of respiratory virus was higher in the fever/RS group than in the non-fever/RS group (44.9% (48/107) versus 23.4% (25/107), p 0.001). Influenza B virus, enterovirus/rhinovirus and coronaviruses were detected more frequently in the fever/RS group, whereas parainfluenza virus 4B and adenovirus were detected more frequently in the non-fever/RS group. Among the non-fever/RS group, chest discomfort was more common among patients tested positive for respiratory viruses than those without respiratory virus detected (44% (11/25) versus 22% (18/82), p 0.04). CONCLUSIONS: Respiratory viruses can be frequently detected among hospitalized patients without typical features of respiratory tract infection. These patients may be a source of nosocomial outbreaks.

Genomic amplification of MET with boundaries within fragile site FRA7G and upregulation of MET pathways in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) is characterized by a poor prognosis making the identification of clinically targetable proteins essential for improving patient outcome. We report the involvement of multiple alterations of the MET pathway in EA development and progression. Microarray analysis of Barrett's metaplasia, dysplasia, and EA revealed overexpression of the MET oncogene in EAs but only those with MET gene amplification. STS-amplification mapping revealed that the boundary of the MET amplicon in these EAs is defined by fragile site FRA7G. We also identified an amplicon at 11p13 that resulted in amplification and overexpression of CD44, a gene involved in MET autophosphorylation upon HGF stimulation. Tissue microarrays with phospho-MET-specific antibodies demonstrated a uniformly high abundance of MET activation in primary EA and cells metastatic to lymph nodes but to a lesser extent in a subset of metaplastic and dysplastic Barrett's samples. Increased expression of multiple genes in the MET pathway associated with invasive growth, for example, many MMPs and osteopontin, also was found in EAs. Treatment of EA-derived cell lines with geldanamycin, an inhibitor for tyrosine kinases including MET receptor kinase, reduced cell migration and induced EA cell apoptosis. The data indicate that upregulation of the MET pathway may contribute to the poor outcome of EA patients and that therapeutic agents targeting this pathway may help improve patient survival.

Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock.

This study was designed to test the hypothesis that tissue factor pathway inhibitor (TFPI) plays a significant role in vivo in regulating coagulation that results from exposure of blood to tissue factor after vascular injury as in the case of gram negative sepsis. Highly purified recombinant TFPI (6 mg/kg) was administered either 30 min or 4 h after the start of a lethal intravenous Escherichia coli infusion in baboons. Early posttreatment of TFPI resulted in (a) permanent seven-day survivors (5/5) with significant improvement in quality of life, while the mean survival time for the controls (5/5) was 39.9 h (no survivors); and (b) significant attenuations of the coagulation response and various measures of cell injury, with significant reductions in pathology observed in E. coli sepsis target organs, including kidneys, adrenals, and lungs. TFPI administration did not affect the reduction in mean systemic arterial pressure, the increases in respiration and heart rate, or temperature changes associated with the bacterial infusion. TFPI treated E. coli infected baboons had significantly lower IL-6 levels than their phosphate buffered saline-treated controls, however tumor necrosis factor levels were similarly elevated in both groups. In contrast to the earlier 30-min treatment, the administration of TFPI at 4 h, i.e., 240 min, after the start of bacterial infusion resulted in prolongation of survival time, with 40% survival rate (2/5) and some attenuation of the coagulopathic response, especially in animals in which fibrinogen levels were above 10% of normal at the time of TFPI administration. Results provide evidence for the significance of tissue factor and tissue factor pathway inhibitor in bacterial sepsis, and suggest a role for blood coagulation in the regulation of the inflammatory response.

Efficacy of treatment with the iron (III) complex of diethylenetriamine pentaacetic acid in mice and primates inoculated with live lethal dose 100 Escherichia coli.

The iron (III) complex of diethylenetriamine pentaacetic acid (DTPA iron [III]) protected mice and baboons from the lethal effects of an infusion with live LD100 Escherichia coli. In mice, optimal results were obtained when DTPA iron (III) was administered two or more hours after infection. Prevention of death occurred in spite of the fact that the adverse effects of TNF-alpha were well underway in the mouse model. The half-life of DTPA iron (III) was 51 +/- 9 min in normal baboons; primary clearance was consistent with glomerular filtration. In septic baboons, survival was observed after administration of two doses of DTPA iron (III) at 2.125 mg/kg, the first one given before, or as late as 2 h after, severe hypotension. Administration of DTPA iron (III) did not alter mean systemic arterial pressure, but did protect baboons in the presence of high levels of TNF-alpha and free radical overproduction. Furthermore, exaggerated production of nitric oxide was attenuated. The mechanism of protection with DTPA iron (III) is not obvious. Because of its ability to interact in vitro with free radicals, its poor cell permeability, and its short half-life, we postulate that DTPA iron (III) and/or its reduced form may have protected the mice and baboons by sequestration and subsequent elimination of free radicals (including nitric oxide) from their systems.

Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts.

Normal cells have a strictly limited growth potential and senesce after a defined number of population doublings (PDs). In contrast, tumor cells often exhibit an apparently unlimited proliferative potential and are termed immortalized. Although spontaneous immortalization of normal human cells in vitro is an extremely rare event, we observed this in fibroblasts from an affected member of a Li-Fraumeni syndrome kindred. The fibroblasts were heterozygous for a p53 mutation and underwent senescence as expected at PD 40. In four separate senescent cultures (A to D), there were cells that eventually recommenced proliferation. This was associated with aneuploidy in all four cultures and either loss (cultures A, C, and D) or mutation (culture B) of the wild-type (wt) p53 allele. Loss of wt p53 function was insufficient for immortalization, since cultures A, B, and D subsequently entered crisis from which they did not escape. Culture C has continued proliferating beyond 400 PDs and thus appears to be immortalized. In contrast to the other cultures, the immortalized cells have no detectable p16INK4 protein. A culture that had a limited extension of proliferative potential exhibited a progressive decrease in telomere length with increasing PD. In the culture that subsequently became immortalized, the same trend occurred until PD 73, after which there was a significant increase in the amount of telomeric DNA, despite the absence of telomerase activity. Immortalization of these cells thus appears to be associated with loss of wt p53 and p16INK4 expression and a novel mechanism for the elongation of telomeres.

A nontransfusional perioperative management regimen for patients with sickle cell disease undergoing laparoscopic cholecystectomy.

BACKGROUND: Patients with sickle cell disease (SCD) are at increased risk for cholelithiasis. Laparoscopic cholecystectomy is the most frequent general surgical operation performed for this group of patients. Acute chest syndrome (ACS) is the most common cause of postoperative death among SCD patients. This study aimed to evaluate the impact of a novel perioperative management regimen involving prophylactic continuous positive airways pressure (CPAP) ventilation and avoidance of preoperative blood transfusion on postoperative SCD-related complications after laparoscopic cholecystectomy. METHODS: A retrospective study included all SCD patients who underwent laparoscopic cholecystectomy since 1997 at our institution. Medical notes were analyzed to assess the rates of postoperative complications in relation to the severity of SCD. RESULTS: A total of 13 patients were identified. There were no recorded episodes of acute painful crises and only one patient experienced an episode of ACS requiring protracted CPAP. CONCLUSION: Laparoscopic cholecystectomy can be safely performed for SCD patients without prior blood transfusion. A defined perioperative regimen including the use of routine postoperative prophylactic CPAP for these patients helps to reduce SCD-related postoperative complications such as ACS and painful vaso-occlusive crises.

Mesenteric oxyhemoglobin desaturation improves with patent ductus arteriosus ligation.

Near-infrared spectroscopy was used to monitor cerebral and mesenteric regional oximetry in a preterm neonate undergoing surgical ligation of a patent ductus arteriosus. This patient initially demonstrated severe mesenteric oxyhemoglobin desaturation, which improved immediately following ductal ligation.

Heavy metal sequential extraction methods--a modification for tropical soils.

Sequential extractions of metals can be useful to study metal distributions in various soil fractions. Although several sequential extraction procedures have been suggested in the literature, most were developed for temperate soils and may not be suitable for tropical soils with high contents of Mn and Fe oxides. The objective of this study was to develop a sequential fractionation procedure for Cu and Zn in tropical soils. Extractions were performed on surface (0-20 cm) samples of ten representative soils of Sao Paulo State, Brazil. Chemically reactive Mn forms were satisfactorily assessed by the new modified procedure. Amorphous and crystalline Fe oxides were more selectively extracted in a new two-step extraction. Soil-born Zn and Cu were primarily associated with recalcitrant soil fractions. The proposed procedure provided more detailed information on metal distribution in tropical soils and better characterization of the various components of the soil matrix. The new procedure is expected to be an important tool for predicting the potential effects of environmental changes and land application of metals on the redistribution of chemical forms of metals in tropical soils.

In vitro transformation of Li-Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.

Transfection of SV40 early region DNA into normal human diploid fibroblasts (NHDFs) increases their proliferative potential to a limited extent. We have investigated the roles of the SV40 large T antigen (LTAg) regions responsible for binding to the protein products of the retinoblastoma (Rb) and p53 genes in this temporary escape from senescence. Patients encoding LTAg mutants were transfected into NHDFs and into Li-Fraumeni syndrome (LFS) fibroblasts which are heterozygous wild-type (wt)/null-mutant for p53. A LTAg mutated in the p53-binding region (T402DE) had greatly reduced efficiency of focus formation, and a p110Rb-binding mutant was unable to induce any foci. T402DE-induced NHDF foci senesced at the same time as untransfected cells, but the equivalent LFS foci all had increased proliferative potentials, with the greatest increase being seen in clones that lost the wt p53 allele. One LFS clone expressed the T402DE mutant during focus formation, but later lost both the T402DE DNA and the wt p53 allele. We conclude that SV40-induced focus formation in NHDFs requires the LTAg p110Rb-binding region, and is enhanced by loss of normal p53 function. In contrast, increased proliferative potential is primarily due to loss of p53 function.

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

Sequence of the cDNA encoding porcine pro-opiomelanocortin.

Messenger RNA was extracted from porcine pituitary pars intermedia and cloned as cDNA by standard methods. The nucleotide sequence encoding porcine pro-opiomelanocortin was established from analysis of two separate cDNA segments having an overlap of 420 bases. The amino acid sequence for the porcine pro-opiomelanocortin protein, which was inferred from the cDNA sequence, was found to correspond exactly to the sequence determined by direct amino acid analysis of the component peptides of pro-opiomelanocortin; namely, the porcine hormones ACTH, beta-lipotropin, gamma-MSH and the connecting peptide. We thus find no evidence for the existence of two porcine pro-opiomelanocortin genes that differ in their coding sequences, as was suggested by the in vitro protein synthesis results of others using mRNA obtained from porcine pituitaries.

Molecular cloning and characterization of a mouse alpha 2C2 adrenoceptor subtype gene.

A clone MA2C2 encoding the murine homologue of adrenoceptor alpha 2C2 was isolated from a mouse genomic library using a subtype specific probe. The nucleotide and the deduced amino acid sequences derived from an ApaI fragment (2 kb) of the clone reveal a single open reading frame encoding a putative receptor protein of 455 amino acids. The 5' untranslated region (0.5 kb) sequenced is characterized by high GC content and CpG island count.

Hypoplastic left heart syndrome: hemodynamic and angiographic assessment after initial reconstructive surgery and relevance to modified Fontan procedure.

After undergoing initial reconstructive surgery for hypoplastic left heart syndrome performed between August 1985 and March 1989, 59 patients (age range 3 to 27 months, mean 13.8 +/- 4.5) underwent elective cardiac catheterization in anticipation of a modified Fontan procedure. Five important hemodynamic and anatomic features considered to be components of successful reconstructive surgery were specifically addressed. 1) Interatrial communication: Only two patients had a measured pressure difference of greater than 4 mm Hg across the atrial septum. 2) Tricuspid valve function: Angiography demonstrated significant tricuspid valve regurgitation in only five patients (moderate in two and severe in three). 3) Aortic arch: Pressure tracings from the right ventricle to the descending aorta revealed a gradient greater than 25 mm Hg in only two patients. 4) Pulmonary vasculature: Ten patients had a calculated pulmonary vascular resistance greater than 4 U.m2; 51 (86%) of the 59 patients had no evidence of distortion (stenosis or hypoplasia) of either the left or the right pulmonary artery. 5) Right ventricular function: Five patients had an end-diastolic pressure in the right ventricle greater than 12 mm Hg and two patients had qualitative assessment of decreased ventricular function. Comparison of catheterization data between survivors and nonsurvivors of the subsequent modified Fontan procedure showed that only significant tricuspid regurgitation is a possible predictor of poor outcome. After first stage reconstructive surgery for hypoplastic left heart syndrome, most survivors have favorable anatomy and hemodynamics at follow-up cardiac catheterization for a subsequent Fontan procedure.

Accuracy of prospective two-dimensional/Doppler echocardiography in the assessment of reparative surgery.

Between January 1987 and January 1989, all 129 patients (aged 11 days to 25 years, median 39 months) undergoing both an echocardiographic examination and cardiac catheterization after reparative surgery were prospectively included in a study to assess the accuracy of combined two-dimensional and Doppler color flow imaging. The patient diagnoses were transposition of the great arteries (n = 20), tetralogy of Fallot (n = 38), coarctation of the aorta (n = 24), complete atrioventricular (AV) canal (n = 15), atrial septal defect (n = 8), ventricular septal defects (n = 3), pulmonary stenosis (n = 4), aortic stenosis (n = 8) and subaortic stenosis (n = 9). In arterial tract stenosis, there was high correlation between Doppler estimates and catheterization-derived measurements of residual right ventricular outflow tract obstruction in patients after the arterial switch operation for transposition of the great arteries (r = 0.95) as well as in patients after corrective repair of tetralogy of Fallot (r = 0.84). In semilunar/AV valve regurgitation, graded as none, mild, moderate or severe, echocardiographic estimates correlated exactly with angiographic grading in 84% and differed by one angiographic grade in the other 16%. In residual left to right shunting, no hemodynamically significant shunt was missed by echocardiography. For residual shunts at the ventricular level (n = 32), addition of Doppler color flow imaging improved the sensitivity (from 63% to 94%) and the negative predictive value (from 88% to 98%). In elevated right ventricular pressure, Doppler-derived right ventricular-right atrial pressure estimates in 24 patients correlated well with catheterization measurements (r = 0.93). Combined two-dimensional and Doppler color flow echocardiography was highly accurate in the prospective evaluation of these four types of postoperative residual.

Mammalian stanniocalcins and cancer.

Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland of bony fish, and is involved in calcium and phosphate homeostasis. The related mammalian proteins, STC1 and STC2, are expressed in a wide variety of tissues. The ovaries have the highest level of STC1, and this increases during pregnancy and lactation. STC1 is present in breast ductal epithelium, and its expression is induced by BRCA1, a tumor suppressor gene that has an important role in breast and ovarian cancer. The expression of STC2 is induced by estrogen, and there is a positive correlation between the level of expression of estrogen receptor and expression of both STC1 and STC2 in breast cancer. This article reviews the data currently available regarding the mammalian STCs, and discusses the roles they may play in normal physiology and in breast and other cancers.

Bleomycin resistance as a selectable marker for transformation of the eukaryote, Dictyostelium discoideum.

An expression cassette was constructed, which has the bacterial bleomycin (Bm) resistance-encoding gene (ble) fused to the Dictyostelium discoideum actin-6 promoter, with a segment of 3'-flanking DNA from the actin-8-encoding gene placed downstream from the ble gene to serve as a transcription terminator. Plasmid pMUW161, which contains this cassette and the D. discoideum plasmid Ddp2 origin of DNA replication, transformed D. discoideum with high efficiency under Bm selection. Hence, this construct is useful as a dominant selectable marker for D. discoideum.