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At least 919 records · Page 51Linked to original sources
PubMed2001

Chlamydia and Borrelia DNA in synovial fluid of patients with early undifferentiated oligoarthritis: results of a prospective study.

OBJECTIVE: More than 50% of patients with synovitis involving 1-4 joints remain classified as having undifferentiated oligoarthritis (UOA) after 1 year of disease. The clinical presentation is often similar to that of reactive arthritis (ReA) and other spondylarthropathies or to Lyme arthritis. We therefore determined how often Chlamydia trachomatis (Ct) and Borrelia burgdorferi (Bb) can be identified in patients with UOA, by using an extensive laboratory approach. METHODS: We prospectively studied 52 patients with UOA who presented at an early synovitis clinic in a region highly endemic for Lyme disease. Patients were examined by standardized clinical and immunoserologic procedures. Synovial fluid was screened for the presence of Ct and Bb DNA by polymerase chain reaction (PCR). Urine was tested for Ct DNA by ligase chain reaction, and serum was tested for Ct antibodies by enzyme-linked immunosorbent assay and Bb antibodies by hemagglutination test and Western blotting. PCR results in the UOA patients were compared with the results in cohorts of patients with definite rheumatoid arthritis (RA), Lyme arthritis, and Chlamydia-induced arthritis (CIA). RESULTS: In the synovial fluid of 9 of 52 patients with UOA (17%), we found Ct DNA, and in 6 of the 52 patients (12%), Bb DNA was found. The frequency of bacteria-specific DNA was 50% (7 of 14) in CIA patients and 69% (11 of 16) in patients with Lyme arthritis. No Bb or Ct DNA was found in the synovial fluid of the 31 RA patients. CONCLUSION: With optimized PCR protocols, it is possible to detect considerable levels of Bb and Ct DNA in the synovial fluid of patients with UOA. Although the presence of bacterial DNA does not unequivocally prove its etiologic significance, we suggest that at least one-third of patients with UOA may have a form of ReA that involves asymptomatic primary infection.

Adolescent↗
PubMed2002

Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae.

As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins.

Cell Fractionation↗
PubMed2007

Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression.

Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.

Capsid Proteins↗
PubMed2007

Characterization of the AlkS/P(alkB)-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations.

The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P(alkB) expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single-cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed-back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned on at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations decreased from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose-limited fed-batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein.

Base Sequence↗
PubMed2004

High-dose methotrexate-induced nephrotoxicity in patients with osteosarcoma.

BACKGROUND: High-dose methotrexate (HDMTX)-induced renal dysfunction can be life threatening, because it delays methotrexate (MTX) excretion, thereby exacerbating the other toxicities of MTX. HDMTX-induced nephrotoxicity has been managed with high-dose leucovorin, dialysis-based methods of MTX removal, thymidine, and with the recombinant enzyme, carboxypeptidase-G2 (CPDG2), which cleaves MTX to inactive metabolites. The objectives of the current study were to estimate the current incidence of HDMTX-induced renal dysfunction in patients with osteosarcoma and to compare the efficacy and recovery of renal function for dialysis-based methods of MTX removal with treatment using CPDG2. METHODS: The literature was reviewed for osteosarcoma trials, use of dialysis-based methods for MTX removal, and reports of MTX-induced nephrotoxicity, including information regarding recovery of renal function. Clinical trial databases of select osteosarcoma studies were reviewed. The efficacy of CPDG2 and renal recovery after CPDG2 rescue was obtained from the database of a compassionate-release trial. RESULTS: Approximately 1.8% of patients with osteosarcoma (68 of 3887 patients) who received HDMTX developed nephrotoxicity Grade >/= 2. The mortality rate among those patients was 4.4% (3 of 68 patients). Dialysis-based methods of MTX removal were used frequently but had limited effectiveness in removing MTX compared with the rapid reductions > 98% in plasma MTX concentrations achieved with CPDG2. CPDG2 did not appear to increase the time to recovery of renal function compared with supportive treatment that included dialysis-based methods. CONCLUSIONS: HDMTX-induced renal dysfunction continues to occur in approximately 1.8% of patients with osteosarcoma who are treated on clinical protocols with optimal supportive care. For patients with delayed MTX excretion and high plasma MTX concentrations, CPDG2 should be considered over hemodialysis to lower plasma MTX concentrations rapidly and efficiently.

Acute Kidney Injury↗
PubMed2001

Prostate biopsy protocols: 3D visualization-based evaluation and clinical correlation.

OBJECTIVES: Systematic needle core biopsy is commonly used for the diagnosis of prostate cancer by urologists worldwide. As accurate and early diagnosis will result in more and better options for treatment, it is critical that the best possible protocols for biopsy be used clinically. In this study, we develop three-dimensional (3D) modeling and simulation technologies to evaluate most of the biopsy protocols in current clinical use, and correlate the results with those from clinical cases. MATERIALS AND METHODS: Using deformable modeling techniques, 3D computerized prostate surface models were reconstructed from step-sectioned, whole-mounted radical prostatectomy specimens with localized prostate cancer. A 3D computer simulation system was developed to accurately depict the anatomy of the prostate and all individual tumor foci. A user-friendly interface was developed in the system so that a physician can easily and interactively use it for prostate needle core biopsy. A total of 281 prostate models were reconstructed, and 18 biopsies were performed by a urologist on each model to determine the detection rates of seven different biopsy protocols. Clinical biopsies from 35 patient cases were also reviewed and correlated with the simulation results. RESULTS: The most commonly used sextant biopsy had only a 71.5% detection rate, while rates for the 10-pattern and 12-pattern protocols were much higher (96.4% and 97.2%, respectively). Even the lateral 4-pattern protocol performed better than the sextant protocol, with a detection rate of 89.3%. The lateral sextant biopsy protocol (using sites similar to, but more lateral than, those in the sextant protocol) achieved a rate of 92.5%. Although the rate of the 14-pattern biopsy was a little higher (97.5%), it used four more biopsies to achieve this increase, which, according to McNemar's test, is not statistically significant when compared to results with the 10-pattern protocol. The 5-region protocol, which uses 12 biopsies, had a detection rate of 89.7%. Transition zone and seminal vesicle biopsies did not result in a significantly increased detection rate when added to the patterns above. The clinical correlation also confirmed that the 10-pattern protocol was significantly superior to the traditional sextant biopsy pattern. CONCLUSIONS: The 10-pattern biopsy protocol was the most optimized among all the protocols evaluated. This protocol supplemented the sextant biopsy protocol with four more lateral biopsies in the mid and apical sites on both sides.

Biopsy, Needle↗
PubMedDate not supplied

Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.

Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were frozen (-20 degrees C) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% +/- 6%, 76% +/- 11%, 92% +/- 6%, respectively) compared with fresh, untreated sperm (19% +/- 16%, P < .05). In addition DNA damage was different (P < .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo-damaged cells in each treatment (r2 = 0.92, P < .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5 degrees C) had a higher percentage of comets than that of fresh sperm (63% +/- 13.9% and 28% +/- 15.6%, respectively, P < .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r2 = 0.805, P < .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.

Animals↗
PubMed2003

Size, shape, and composition of wear particles from metal-metal hip simulator testing: effects of alloy and number of loading cycles.

There has been a revived interest in metal-metal total hip replacements because of their potential for improved wear performance compared with conventional metal-polyethylene implants. The aim of the present study was to characterize metal wear particles isolated from metal-metal hip simulator testing of various clinically relevant alloys and to analyze the effects of these alloys and the number of loading cycles on wear particle characteristics. Implants were manufactured using medical-grade cobalt-chromium-molybdenum (CoCrMo) alloys that were high-carbon wrought, low-carbon wrought, or cast (with solution annealing). Testing was performed in a MATCO orbital bearing hip simulator in 95% bovine calf serum. The wear particles were isolated from the serum at test periods of 0-0.25 million cycles (Mc) (run-in wear) and 1.75-2 Mc (steady-state wear) using an enzymatic protocol previously optimized to minimize particle changes due to reagents. Isolated particles embedded in epoxy resin were characterized by transmission electron microscopy (TEM) and energy dispersive X-ray analysis (EDXA). The EDXA results revealed the predominance of "lighter" particles containing Cr and O (most likely chromium oxide particles from the passivation layer) and fewer darker CoCrMo particles, with varying ratios of Co and Cr (possibly from carbides and from implant matrix material). More CoCrMo particles were observed with the low-carbon wrought alloy, but the majority of the particles for all three alloys was chromium oxides, especially for the 1.75-2 Mc test period. Image analysis of TEM micrographs revealed that for 0-0.25 Mc, there was up to 21% needle-shaped particles but that the majority remained round to oval in shape, reflecting the predominance of chromium oxide particles. Particle length averaged about 52 +/- 4 nm, with only small differences due to the alloy. For 1.75-2 Mc, most particles were round to oval in shape. They were even less needle-shaped than at 0.25 Mc, and they had a slightly smaller length, averaging 46 +/- 3 nm. In addition to characterizing the size and shape of particles from a MATCO simulator, this study is the first to demonstrate that particles that do not contain Co (presumably chromium oxides) can be predominant in the wear of metal-metal hip implants. It is therefore recommended that future in vitro and in vivo studies include the effects of these particles rather than just the effects of CoCrMo particles on the overall tissue response.

Alloys↗
PubMed2003

Specific empirical free energy function for automated docking of carbohydrates to proteins.

We present an automated docking protocol specifically optimized to predict the structure and affinity of a protein-carbohydrate complex. A scoring function was developed based on a training set of 30 protein-carbohydrate complexes of known structure and affinity. Combinations of several models for hydrogen bonding, torsional entropy loss, and solvation were tested for their ability to fit the training set data, and the best model was used with AutoDock. The electrostatic empirical coefficient is larger than in a previously obtained model using a training set comprised of various types of protein-ligand complexes, indicating that electrostatic interactions play a more important role in determining the affinity between a carbohydrate and a protein. The differences in the relative weighting of the empirical coefficients in the model yields predicted free energies for the training set with a standard error of 1.403 kcal/mol. The new scoring function was tested on 17 Aspergillus niger glucoamylase inhibitors for which binding energies had been determined experimentally. Free energies of complex formation were predicted with a residual standard error of 1.101 kcal/mol. The new scoring function therefore provides a robust method for predicting free energies of formation and optimal conformations of carbohydrate-protein complexes.

Algorithms↗
PubMed2003

Resolving morphology and antibody labeling over large distances in tissue sections.

Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.

Animals↗
PubMed1994

Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy.

The detection of elemental distributions within ultrastructural cellular components presents a number of challenges. There are many technical questions that need to be resolved including optimal fixation protocols. Another is the impact of heavy metals, such as osmium tetroxide (OsO4), on the detectability of other elements when OsO4 is used in chemical fixation protocols for biological samples. OsO4 was examined by varying its concentrations from 0% to 1% and time of fixation from 5 to 30 minutes with hamster alveolar macrophages. The morphological quality of cellular images observed and the detectability of iron using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) were evaluated. One percent OsO4 for 30 minutes in the chemical fixation protocol enhances the quality of the ESI and does not interfere with the ESI or EELS signal of iron. Positive results from both methods indicate the presence of the specific element. The loss of 59Fe during the chemical fixation procedure was also studied. Less than 10% was lost during the primary fixation step, but minimal losses occurred through dehydration, embedding, and sectioning. Careful technical assessment of the presence of an element as well as factors which might interfere with its detection is an important step in the application of any analytical microscopic technique.

Animals↗
PubMed2004

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.

Base Sequence↗
PubMed2001

Contrast-enhanced coronary MR angiography: relationship between coronary artery delineation and blood T1.

Contrast-enhanced coronary angiography has become an important technique for magnetic resonance (MR) coronary artery imaging. However, the relationship between the quality of the coronary artery images and blood T1 has not yet been fully explored. In this paper, we assessed this relationship in an animal model by using a prototypical blood pool agent. With accumulated injections of this agent, the blood T1 would be maintained at different levels. The measured blood T1 values in vivo were 147 +/- 3, 82 +/- 6, 48 +/- 4, 40 +/- 3, and 30 +/- 8 msec (N = 7). Fixed and variable flip angle schemes were used in coronary artery imaging. The signal to noise ratios (SNR) of coronary arteries were measured and the image quality was assessed. It was found that blood T1 less than 80 msec might be desired. No statistically significant difference was observed between two flip angle schemes. There was better vessel definition using variable flip angle at blood T1 lower than 50 msec. Understanding this relationship may be beneficial to optimizing image protocol and/or design of blood pool contrast agents for contrast-enhanced coronary angiography.

Animals↗
PubMed2006

Scoutless stepping-table peripheral contrast-enhanced MR angiography.

PURPOSE: To evaluate the feasibility of a scoutless method, termed EZ-STEP, for stepping-table peripheral contrast-enhanced magnetic resonance angiography (CE-MRA). MATERIALS AND METHODS: This scoutless method involves the use of a stepping-table, fast 3D MRA acquisition that incorporates spatially nonselective radiofrequency (RF) pulses for excitation to reduce the repetition time (TR). The sequence was tested in a phantom. The EZ-STEP protocol was optimized in four healthy volunteers and used in 15 subjects. The image quality was scored in a blinded fashion and compared with conventional MRA in eight patients. RESULTS: The acquisition speed of the EZ-STEP sequence was approximately 30% faster in the phantom study compared to the conventional MRA sequence. The total examination time for EZ-STEP was 6 minutes, compared to an average of 23 minutes for conventional MRA. The average image quality scores for EZ-STEP and conventional MRA for stations 1-3 were 3.50 vs. 3.06 (P = 0.087), 3.53 vs. 3.00 (P = 0.033), and 2.97 vs. 2.50 (P = 0.090), respectively. CONCLUSION: EZ-STEP is a more efficient method than the conventional approach for stepping-table peripheral CE-MRA, and provides comparable or better image quality. This method shortens the examination time substantially and eliminates the risk of failing to image a vessel because of improper positioning of the scan volume.

Adult↗
PubMed2002

Diagnosis and quantitative evaluation of parvovirus B19 infections by real-time PCR in the clinical laboratory.

A real-time PCR assay was developed for quantitative detection of B19 DNA in clinical serum samples. The assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. With an optimized PCR protocol, this system was able to quantitate the target DNA down to 3 x 10(1) genome copies/reaction and to detect as few as 3 x 10(0) genome copies/reaction. Real-time PCR was used to detect B19 DNA in 108 serum samples from patients with a clinical suspicion of B19 infection, showing a sensitivity of 92.7% and a specificity of 100% when compared with a standardized PCR-ELISA considered as the standard. Using the LightCycler assay, the entire procedure of detection and quantitation of B19 DNA in clinical serum samples took up to 90 min proving five times faster than PCR-ELISA. B19 DNA quantitation in positive samples by real-time PCR showed a mean of 1.1 x 10(9) B19 DNA copies/ml in samples in the acute active phase of B19 infection (DNA+, IgM+, IgG-), 4.3 x 10(6) B19 DNA copies/ml in samples in the active phase (DNA+, IgM+, IgG+), 3.7 x 10(5) genome copies/ml in samples in the long-lasting active phase (DNA+, IgM-, IgG+) with a statistically significant reduction of B19 DNA content between the group of sera in the acute active phase and the group of sera in the active phase of B19 infection. The high levels of sensitivity, specificity, and rapidity provided by the LightCycler technology for the detection and quantitation of B19 DNA represent a significant improvement for the laboratory diagnosis of B19 infection.

DNA, Viral↗
PubMed2006

Age-dependent changes in matrix composition and organization at the ligament-to-bone insertion.

Injuries to the anterior cruciate ligament (ACL) often occur at the ligament-to-bone insertion site; thus, an in-depth understanding of the native insertion is critical in identifying the etiology of failure and devising optimal treatment protocols for ACL injuries. The objective of this study is to conduct a systematic characterization of the ACL-to-bone interface, focusing on structural and compositional changes as a function of age. Using a bovine model, three age groups were studied: Neonatal (1-7 days old), Immature (2-6 months old), and Mature (2-5 years old). The distribution of types I, II, X collagen, decorin, cartilage oligomeric matrix protein (COMP), glycosaminoglycan (GAG), alkaline phosphatase (ALP) activity, and minerals at the ACL-to-bone insertion were examined. Additionally, cell aspect ratio, size, and distribution across the insertion were quantified. The ACL-to-bone insertion is divided into four regions: ligament, nonmineralized interface, mineralized interface, and bone. Both region-dependent and age-dependent structural and compositional changes at the insertion site were observed in this study. The interface in the skeletally immature group resembled articular cartilage, while the adult interface was similar to fibrocartilaginous tissue. Age-dependent changes in extracellular matrix composition (type X collagen, sulfated glycosaminoglycan), cellularity, ALP activity, and mineral distribution were also found. Marked differences in collagen fiber orientation between the femoral and tibial insertions were observed, and these differences became more pronounced with age.

Aging↗
PubMed2004

Pathology of melanocytic lesions: new, controversial, and clinically important issues.

Patients with primary cutaneous melanocytic lesions rely not only on the knowledge, skills, and experience of their treating clinician but also on the fundamentally important input of their pathologist for accurate diagnosis and appropriate management. Free and precise communication between pathologists and surgeons is important and undoubtedly improves patient care, particularly when managing difficult or complicated cases. To provide both patient and surgeon with the necessary information they require to make the most appropriate decisions, the pathology report should include all pathologic factors that are important in determining the patient's prognosis and management. Use of a synoptic format for pathology reporting of melanomas can facilitate this. Recent studies have established that the dermal mitotic rate of a primary cutaneous melanoma is a major prognostic determinant, and have shown that its assessment and that of other important histopathologic prognostic variables are reproducible between pathologists. Sentinel node (SN) biopsy has provided a minimally invasive procedure that can accurately predict the regional node status of melanoma patients. It is well demonstrated that the use of immunohistochemical stains assists in the detection of melanoma micrometastases in SNs, although it remains unclear which is the optimal pathologic protocol for SN evaluation and whether there is a role for reverse transcriptase polymerase chain reaction (RT-PCR) in SN assessment. False negative SN biopsies may occur as a result of errors in lymphatic mapping or sentinel lymphadenectomy, or because of a deficiency in the process of histopathologic evaluation. Recent studies have shown that the likelihood of non-SN involvement when the SN is positive correlates mostly with the extent of SN involvement, in particular the tumor penetrative depth (defined as the maximum distance of melanoma cells from the inner margin of the SN capsule). It appears that assessment of the micromorphometric features of positive SNs may be useful in predicting which patients have a low probability of having metastatic tumor in non-SNs, and therefore in selecting patients who potentially may be spared a completion lymph node dissection. It is likely that future advances in our understanding of the molecular biology of melanoma will provide new insights into tumor classification, improve diagnostic accuracy and prognostic ability, and lead to the development of more precisely targeted therapies.

Biopsy, Fine-Needle↗
PubMed2005

Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction.

In the present work, a rapid, accurate, and sensitive method has been developed for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in edible animal tissues (muscle tissue, liver, kidney, and eggs). The separation was accomplished on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature within 15 min. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v). UV detection at 275 nm yielded the following limits of detection: 100 pg per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 20 pg for ofloxacin, and 200 pg for enrofloxacin. Peaks in real samples were identified by means of a photodiode array detector. The method was validated in terms of intra-day (n = 8) and inter-day (n = 8) precision and accuracy. Tissue samples were purified from endogenous interference by solid-phase extraction using Oasis HLB cartridges. The solid-phase extraction protocol was optimized in terms of retention and elution. Recovery rates at fortification levels of 40, 60, and 80 ng/g ranged from 82.5% to 111.1%. The applicability of the method was examined using real samples from a chicken treated orally with the five studied fluoroquinolones.

Animals↗