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PubMed2006

The effect of bone tunnel dilation versus extraction drilling on the initial fixation strength of press-fit anterior cruciate ligament reconstruction.

INTRODUCTION: Serial dilation of the bone tunnel has been reported to create a tighter graft-tunnel fit. It was hypothesized that a serial dilation of the femoral bone tunnel would increase the initial fixation strength in press-fit anterior cruciate ligament (ACL) reconstructive surgery. MATERIALS AND METHODS: Initial fixation strength of the femoral press-fit fixation technique was investigated in 72 porcine specimens in an ex vivo study by varying the femoral tunnel preparation technique. Extraction-drilling, tunnel dilation by 1 mm and dilation by 2.5 mm were assessed. Initial fixation strength of press-fit fixated patellar tendon-bone grafts was tested within each preparation group conducting a single cycle (and cyclic) load to failure protocol. The resulting tunnel diameter and the porcine femoral bone mineral density were determined using microradiographs and peripheral quantitative CT scans, respectively. RESULTS: Dilating a previously extraction-drilled femoral bone tunnel by 1 mm significantly enhances initial press-fit fixation strength in both single cycle and cyclic load to failure testing when compared to extraction-drilling and tunnel dilation by 2.5 mm. Due to an initial spring-back effect the resulting diameter of the femoral tunnel was underestimated by 3.3% with drilling and 6.7 and 12.2% with dilation by 1 and 2.5 mm, respectively. Volumetric trabecular bone mineral density at the site corresponding to the area of tunnel placement averaged 318 mg/cm(3). CONCLUSION: Dilating a femoral tunnel that is underdrilled by 1 mm appears to be a reasonable technical procedure in order to enhance initial fixation strength of press-fit ACL graft fixation.

Analysis of Variance↗
PubMed2006

[Optimisation of retrospective diagnosis of cytomegalovirus congenital infection from dried blood spots].

Out of the 90% of cytomegalovirus (CMV) congenitally infected children that are asymptomatic at birth, 5 to 15% will later develop complications, mainly neurodevelopmental defects and/or deafness. Unfortunately, after the first 2 weeks of life, usual diagnostic techniques for CMV detection (viral culture and serology) are useless to differentiate congenital infection from post-natal acquired infection, whereas detection of viral DNA from dried blood spots (DBS; Guthrie cards), systematically collected from all newborns in the first days of life, has been described for late diagnosis of CMV congenital infection. The aim of our study was to choose and optimise a viral DNA extraction method from DBS and to study if CMV DNA detection is reliable when DBS are stored for 1 year at room temperature or 2 months at 37 degrees C. 10 reference cards (blood collected from CMV seronegative newborns (IgG/IgM negative) were "infected" with serial dilutions of virus and spotted on Guthrie cards) were tested. 3 extraction methods were evaluated, products of PCR were analyzes by agarose gel electrophoresis and quantification of CMV from DBS was also performed. Analysis of the results obtained from reference cards showed higher sensitivity of phenol/chloroform extraction following treatment with proteinase K, compared to heat extraction in cell culture medium or extraction with a commercial kit. We did not observe quantitative loss of viral DNA after 1 year storage at room temperature. CMV DNA detection from Guthrie cards could become a very useful tool for retrospective diagnosis of congenital CMV infection when sequelae are diagnosed in the first years of life. We are pursuing this study with DBS from congenitally infected children.

Cytomegalovirus↗
PubMed1991

[In vitro cytotoxicity of Ganoderma lucidum on oral cancer cells].

The extract from the mycelium of Ganoderma lucidum was diluted into serial concentrations and added into in vitro cultured oral cancer and normal cell lines. After incubation for 24 hours, the survival fraction was determined by MTT colorimetric assay. The result revealed that the ID50 was about 3mg/ml and the total lethal dosage was beyond 4 mg/ml. This toxic effect was the same in both cancer and normal cells. Not only was there no difference between cancer and normal cells, but also the high dosage required in toxicity leads to the conclusion that the GL has no direct cytotoxic effect in cancer treatment.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.

Bacterial Proteins↗
PubMed2000

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR.

Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used. Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration. Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone. PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection. It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies.

Animals↗
PubMed1996

Peripheral nerve extract effects on mesenchymal cells.

Several common congenital limb disorders are characterized by normal tissue differentiation but abnormal somatic growth. These include: idiopathic clubfoot, idiopathic leg length discrepancy, hemi-atrophy and hemi-hypertrophy. Both clinical and research studies have suggested that peripheral nerves may be important in regulating somatic growth of limb tissues. To investigate the hypothesis that peripheral nerves convey trophic substances to mesenchymal tissues that are involved in the regulation of growth, we developed an in vitro assay to assess the effect of fractions of peripheral nerve on myoblast and chondroblast growth and differentiation in a mammalian (rat) system. Whole rat sciatic nerve extract was fractionated by ammonium sulfate precipitation and by affinity chromatography. Concavalin A chromatography resolved whole nerve extract into a glycoprotein and a non-glycoprotein fraction. Serial ammonium sulfate precipitation yielded three pellet fractions designated as 35%, 70%, and 100% pellets; corresponding to ammonium sulfate concentrations of 0 to 35%, 35 to 70%, and 70 to 100% saturation, respectively. Dialyzed solutions of these pellets as well as the fractions from Concavalin A chromatography were assayed for biological activity in micromass cultures of rat limb bud mesenchyme, which allowed assessment of both myoblast and chondroblast stimulation. Stimulation of protein synthesis and myoblast proliferation (as measured by MF20 staining) occurred with both 70% and 100% ammonium sulfate fractions. Stimulation of chondroblasts (as measured by the number of alcian blue staining nodules) occurred with the 35% and 100% fractions. The glycoprotein fraction from the affinity chromatography stimulated protein synthesis and myoblast proliferation and inhibited chondroblast development. Stimulation of chondroblasts was seen with the non-glycoprotein fraction. No effect on protein synthesis, myoblast proliferation or chondroblast proliferation was found in cultures treated with rat transferrin (transferrin has been reported to stimulate myoblasts in avian culture systems).

Ammonium Sulfate↗
PubMed2001

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Animals↗
PubMed1994

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid.

Animals↗
PubMed1986

Growth responses of normal, benign hyperplastic, and malignant human prostatic epithelial cells in vitro to cholera toxin, pituitary extract, and hydrocortisone.

Normal, benign hyperplastic (BPH), and malignant prostatic epithelial cells from adult humans can be serially passaged in medium PFMR-4 supplemented with 20% whole serum, cholera toxin, and epidermal growth factor (EGF). Improved growth of these cells has been achieved by optimizing the concentrations of the components of the basal medium. In the new modified medium, PFMR-4A, the only supplements required are 5% dialyzed serum, cholera toxin, EGF, pituitary extract, and hydrocortisone. Comparative studies of the growth responses of prostatic cells derived from normal central zone, normal peripheral zone, BPH nodules, and adenocarcinomas did not reveal any qualitative differences; all respond positively to the addition of cholera toxin, pituitary extract, and hydrocortisone to the culture medium. Cells of one cancer strain, however, were much less density-dependent for growth than were the other strains of normal, BPH, or malignant cells.

Adult↗
PubMedDate not supplied

[Human milk and very low birth weight nutrition].

INTRODUCTION: Enteral feeding strategies of very low birth weight (VLBW) infants and when to start them have changed significantly in the last few years. Controversy exists on which is the best regimen to feed this high risk group, since human milk has insufficient quantities of some nutrients. Fortification of human milk improves growth rates and maintains immunologic, metabolic and emotional benefits. OBJECTIVE: To examine if early feeding of VLBW infants with higher amounts of human milk and human milk fortifiers could improve post-natal growth and maintain human milk production. STUDY DESIGN: Prospective study with historical control. SUBJECTS AND METHODS: A group of 100 VLBW infants that were born between September 1999 and April 2000 were studied and were compared with another randomized group of similar birth weight (n = 31) that was born previously. The studied group was fed early with human milk (enteral trophic nutrition) and fortified with human milk fortifier when 100 mL/Kg/day were reached, with rapid increase of the supplies according to tolerance and hemodynamic state. The control group was not fed early with human milk and was not given human milk fortifier. The collection of human milk in the studied group was highly stimulated. Mothers extracted their milk more frequently in the "Human Milk Lactary" and lived in a "Residence for Mothers" while their neonates were in the hospital. The mothers who could travel to the hospital were allowed to extract their milk in their homes with a supervised and controlled method (serial bacteriology, "Lactary 24 Hours Program"). RESULTS: Early feeding with human milk (enteral trophic nutrition), higher amounts of human milk and fortified with human milk fortifier, improved enteral tolerance and diminished significantly the number of days needed to regain birth weight, the days of fast and the days to reach the total enteral supply. Frequent extractions of human milk increased the available amounts of milk and allowed relactation. At discharge the infants were fed on breast-milk and formulas and their growth curves were adequate at their first year of post conception age. CONCLUSION: On stable conditions, very low birth infants soon after birth, should be fed with a combination of human milk and human milk fortifiers. Our study demonstrated an improved infant post-natal growth while mothers' milk production was maintained. COMMENTS: To feed human milk with human milk fortifiers to this high risk infant group, the neonatal health team must be highly motivated and committed to encourage mothers to express their milk. This also encourages breast feeding during the subsequent months. In Latin America communities, continuance of breast feeding for longer periods of time is a public health priority.

Food, Fortified↗
PubMed2002

Plasma proteins in a standardised skin mini-erosion (II): effects of extraction pressure.

BACKGROUND: A standardised suction technique has been used to sample plasma proteins in dermal interstitial fluid (IF) serially for 5 to 6 days from a suction-induced skin mini-erosion. Increased protein concentrations ascribed to inflammation have been shown from day 1 onward. In this study, we assessed the effect of two different extraction pressures on IF sample composition. METHODS: Total protein concentration and the concentrations of insulin, prealbumin, albumin, transferrin, IgG and alpha-2-macroglobulin were assessed daily in healthy volunteers. Samples were extracted at 50 mmHg and 200 mmHg below the atmospheric. RESULTS: At 0 h after forming the erosion, mean total IF protein content (relative to plasma) was lower in the samples extracted at -200 mmHg than at -50 mmHg (26 +/-13% (SD) vs 48 +/-9.8%; p < 0.05). There were no significant differences at 24, 48, 72 or 96 h. Of the individual proteins, expressed as area units (AU) for area under the curve (AUC) from 0-96 h, albumin was lower in IF sampled at -200 mmHg (2.49 +/- 0.68 vs 3.08 +/- 0.36 AU; p < 0.05), as was transferrin (1.91 +/- 0.52 vs 2.40 +/- 0.42 AU; p < 0.05). Extraction volumes were significantly higher at -200 mmHg (AUC diff: 60%; p < 0.05). CONCLUSIONS: Samples of IF extracted at 0 h at -200 mmHg contained lower protein concentrations, indicating an increased water fraction and an intact sieve function of the vascular wall. The difference in protein concentration extracted at higher and lower pressure from 24 h onward was less pronounced. Lower pressure should be used to sample substances of greater molecular size.

Blood Proteins↗
PubMedDate not supplied

A method for measurement of cross sectional area, segment length, and branching angle of airway tree structures in situ.

Accurate quantitative measurements of airway and vascular dimensions are essential for evaluating function in both the normal and in the diseased lung. This report describes a new integrated method for three-dimensional (3D) extraction and analysis of pulmonary tree structures using data from High Resolution Computed Tomography (HRCT). Serially scanned two-dimensional (2D) slices of the lower left lobe of isolated dog lungs were stacked to create a volume of data. Airway and vascular trees were extracted using a 3D seeded region-growing algorithm based on differences in CT number between wall and lumen. In the region-growing step, voxels in the lumen are tagged with a distance descriptor to identify points along the tree structure equidistant from the seed point. To obtain quantitative data, we reduced each tree to its central axis. From the central axis, branch length was measured as the distance between two successive branch points, branch angle was measured as the angle produced by two daughter branches, and cross-sectional area was measured from a plane perpendicular to the central axis point. Data derived from these methods can be used to localize and quantify structural differences both during different physiologic conditions and in pathologic lungs.

Algorithms↗
PubMed1976

Sauna-takers disease. Hypersensitivity pneumonitis due to contaminated water in a home sauna.

Daily saunas taken by a young man were followed by fever, chills, malaise, dyspnea, cough, and myalgia from six to eight hours later. Symptoms, which were related to pouring water from a sauna bucket over the heating element, progressed to chronic dyspnea and fatigue. Serial serum samples showed precipitin reactions to bucket water and extracts of bucket mold. IgG antibody activity, demonstrated by radioimmunoassay, suggested that Pullularia was a major antigen.

Adult↗
PubMed1978

Further characterization of CSAp, an antigen associated with gastrointestinal and ovarian tumors.

Colon-specific antigen-p, or CSAp, was originally extracted from GW-39 tumors, which are human colonic carcinomas serially transplanted in golden hamsters, and antibodies to CSAp have been produced in the same animal hosts. By means of immunodiffusion and a hemagglutination-inhibition assay, CSAp has been found to be restricted to adult and fetal small intestine, neoplastic gastric and colonic tissues, inflamed colon, and cystic mucinous tumors of the ovary. CSAp was shown to be distinct from blood group antigens, including Lea and Leb blood group substances, liver ferritin, AFP, CEA, CSA, CMA, ZGM, and BOFA, and to have the electrophoretic mobility of an alpha2-globulin. Gel filtration studies indicated that CSAp in GW-39 tumor, primary human colonic carcinoma, and ovarian cancer mucinous cyst fluid had a peak molecular size range of 70,000--110,000. Quantitation of CSAp in 214 tissue specimens by the hemagglutination-inhibition assay revealed a progressive increase in fetal, inflamed, and neoplastic intestine, such that CSAp in colonic tumors was increased over normal colon tissue. Thus, CSAp appears to be an organ-specific antigen showing increased levels in some gastrointestinal and ovarian neoplasms, as well as in specimens with colitis.

Alpha-Globulins↗
PubMed2001

Cytokine pattern in aneurysmal and occlusive disease of the aorta.

BACKGROUND: Prominent inflammatory infiltrates of macrophages and T-lymphocytes are found in both aortic occlusive disease (AOD) and abdominal aortic aneurysms (AAA). These cells secrete different cytokines that might affect matrix turnover through modulation of matrix metalloproteinase expression. A different cytokine pattern might account for the evolution of AOD vs AAA. MATERIALS AND METHODS: Six different cytokines were examined to determine whether AOD and AAA could be characterized by unique cytokine patterns. AOD (n = 8) and AAA (n = 8) tissues were collected and serially treated with salt, dimethyl sulfoxide, and urea buffers to extract the soluble matrix or cell-bound cytokines. Levels of IL-1 beta, TNF-alpha, IL-10, IL-12, and IFN-gamma were measured by immunoenzymatic methods. Additionally, RNA levels of IL-12 and IFN-gamma were measured. RESULTS: AAA tissue contained higher levels of IL-10 compared to AOD tissue (P < 0.05). Higher levels of the proinflammatory cytokines IL-1 beta, TNF-alpha, and IL-6 were found in AOD (P < 0.05). mRNA levels of IL-12 and IFN-gamma did not differ between the diseases. Aortic tissues contained large amounts of matrix or cell-bound cytokines. CONCLUSIONS: AAA is characterized by greater levels of IL-10 while IL-1 beta, TNF-alpha, and IL-6 are higher in AOD. Targeted deletion of these cytokines in animal models might help in identifying their role in the progression of AAA.

Aortic Aneurysm, Abdominal↗
PubMed1993

An integrated approach to biodistribution radiation absorbed dose estimates.

An integrated approach to existing methods of extracting biodistribution data, pharmacokinetics and radiation absorbed dose estimates from serial scintigraphic images is described. This approach employs a single computer-generated user interface to reformat planar scans into a standard file type, align conjugate (anterior and posterior) images, draw regions of interest (ROIs) over selected organs and lesions and generate count data for anterior and posterior views and calculated geometric means. Using standard correction methods, the fraction injected activity is obtained for all ROIs and total body. This methodology has been applied to the analysis of indium-III-labelled breast-cancer-directed antibodies and technetium-90m-labelled CEA-specific antibody fragments in non-small-cell lung cancer. It is anticipated that this approach will be useful for evaluating the dosimetry of other radiolabelled monoclonal antibodies, as well as other radiopharmaceuticals.

Breast Neoplasms↗
PubMed1993

Physiological modelling of renal drug clearance.

A physiological model of renal drug clearance is presented with the aim of establishing a basis for adjusting drug dosing regimens in renal insufficiency. In agreement with the morphology of blood supply to the nephron, the model assumes serial arrangement of the processes involved in drug excretion. Fractional extraction by filtration in the glomeruli is defined in terms of the product of the unbound fraction of the drug, the filtration fraction being responsible for the limited extraction efficiency of this process. For a description of the limitations of the tubular secretory process by plasma flow through peritubular capillaries, the parallel tube model is utilized. The assumption of direct proportionality between the transport maximum of the secretory process and filtrate flow in the tubules permits a quantitative comparison of the intrinsic tubular secretion clearance and the effectiveness of the filtration process. Provided that the secretory mechanism is highly effective, renal clearance becomes dependent only on kidney plasma flow and the fraction of drug not reabsorbed in the tubules. Tubular reabsorption results only in a proportional decrease in renal clearance. The model predicts proportionality of renal drug clearance to GFR, which as a rule is used for dosage adjustment of drugs in renal insufficiency, only for compounds exclusively excreted by filtration. Compounds also excreted by tubular secretion in general exhibit a curvilinear relationship. The curvature is less pronounced as an increasing fraction of the drug is protein bound in blood. Therefore, for dosage adjustment of drugs secreted in the tubules and highly bound in blood, proportionality between renal clearance and GFR can serve as a reasonable approximation.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerular Filtration Rate↗
PubMed2005

A robust method for alignment of histological images.

Determination of reference points is a precondition for reconstruction of serial sections. In the case of comprehensive reconstruction work, manual extraction of the markers may be very time-consuming and may even make such reconstruction impossible. The procedure presented in this contribution allows automatic alignment of histological preparations provided that nuclei or comparable structures extend over several sections and are capable of being extracted using methods of pattern recognition. The method was applied to 50 sections with Nissl staining. The nuclei were extracted from the images and evaluated by application of the algorithm. All image pairs were correctly aligned. An integrated control mechanism ensures automatic detection of incorrectly aligned images.

Algorithms↗