Long-term outcome of lymph vessel transplantation after chronic lymphorrhea.

A 52-year-old male patient developed a chronic fistula with excessive lymph leakage in the left axilla following removal of an enlarged lymph node with chronic local adipose tissue inflammation due to infection. After multiple surgeries, treatment with lymphatic vessel transplantation was successful. No recurrence occurred over 20 years of follow-up.

IgE responses to multiple allergen components among school-aged children in a general population birth cohort in Tokyo.

BACKGROUND: Response patterns to allergen components among Japanese children have not been studied extensively. OBJECTIVE: Our aim was to examine the differences in sensitization patterns at ages 5 years and 9 years to identify longitudinal changes in the degree and patterns of sensitization in a birth cohort of Japanese children. METHODS: Our study enrolled 984 children at aged 5 years between 2008 and 2010, and 729 children aged 9 years between 2012 and 2014. Allergic diseases were assessed using the ISAAC and UK Working Party's Diagnostic Criteria. Serum-specific IgE titers to allergen components were measured by multiplex array ImmunoCAP ISAC when the children were aged 5 and 9 years. Principal component analysis (PCA) was performed to characterize IgE sensitization to allergen components. RESULTS: The prevalence of allergic rhinitis increased considerably over time (10.6%-31.2%). Furthermore, the sensitization prevalence to allergen-specific IgE (sIgE) also increased from 57.8% at age 5 years to 74.8% at age 9 years. IgE sensitization prevalence to Der f 1 (mites) was 42.1% at age 5 years and 54.3% at age 9 years. Furthermore, children were highly sensitized to Cry j 1 (Japanese cedar) (32.8% at age 5 years and 57.8% at age 9 years). Principle component analysis showed that sensitization to PR-10 cross-reactive components was independent of sensitization to mite and that no children acquired sensitization to pollen before acquiring sensitization to mite. CONCLUSIONS: The prevalence of allergic rhinitis and related allergen components increased from age 5 years to age 9 years in Japanese children.

Effect of Sudden Deprivation of Sensory Inputs From Periodontium on Mastication.

OBJECTIVE: To investigate the effect of sudden deprivation of sensory inputs from the periodontium on jaw kinematics and time-varying activation profile of the masseter muscle. METHODS: Fourteen (age range: 22-26 years; four men) healthy and natural dentate volunteers participated in a single experimental session. During the experiment, the participants were asked to eat six hard visco-elastic test food models, three each before and after an anesthetic intervention. The movements of the jaw in three dimensions and electromyographic (EMG) activity of the masseter muscle on the chewing side were recorded. RESULTS: The results of the study showed no significant differences in the number of chewing cycles (P = 0.233) and the duration of chewing sequence (P = 0.198) due to sudden deprivation of sensory inputs from the periodontium. However, there was a significant increase in the jaw opening velocity (P = 0.030) and a significant increase in the duration of occlusal phase (P = 0.004) during the anesthetized condition. The EMG activity of the jaw closing phase was significantly higher during the control condition [116.5 arbitrary units (AU)] than anesthetized condition (93.9 AU). The temporal profile of the masseter muscle showed a biphasic increase in the excitatory muscle drive in the control condition but this increase was virtually absent during the anesthetized condition. CONCLUSION: Sudden deprivation of sensory inputs from the periodontium affects the jaw kinematics and jaw muscle activity, with a clear difference in the time-varying activation profile of the masseter muscle. The activation profile of the masseter muscle shows that periodontal mechanoreceptors contribute to approximately 20% of the EMG activity during the jaw closing phase.

High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome.

The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches ≥200 × 106 cells mL-1 . The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production.

Non-targeted analysis of unexpected food contaminants using LC-HRMS.

A non-target analysis method for unexpected contaminants in food is described. Many current methods referred to as "non-target" are capable of detecting hundreds or even thousands of contaminants. However, they will typically still miss all other possible contaminants. Instead, a metabolomics approach might be used to obtain "true non-target" analysis. In the present work, such a method was optimized for improved detection capability at low concentrations. The method was evaluated using 19 chemically diverse model compounds spiked into milk samples to mimic unknown contamination. Other milk samples were used as reference samples. All samples were analyzed with UHPLC-TOF-MS (ultra-high-performance liquid chromatography time-of-flight mass spectrometry), using reversed-phase chromatography and electrospray ionization in positive mode. Data evaluation was performed by the software TracMass 2. No target lists of specific compounds were used to search for the contaminants. Instead, the software was used to sort out all features only occurring in the spiked sample data, i.e., the workflow resembled a metabolomics approach. Procedures for chemical identification of peaks were outside the scope of the study. Method, study design, and settings in the software were optimized to minimize manual evaluation and faulty or irrelevant hits and to maximize hit rate of the spiked compounds. A practical detection limit was established at 25 µg/kg. At this concentration, most compounds (17 out of 19) were detected as intact precursor ions, as fragments or as adducts. Only 2 irrelevant hits, probably natural compounds, were obtained. Limitations and possible practical use of the approach are discussed.

Suspect screening of OH-PAHs and non-target screening of other organic compounds in wood smoke particles using HR-Orbitrap-MS.

Wood combustion has been shown to contribute significantly to emissions of polycyclic aromatic hydrocarbons and hydroxylated polycyclic aromatic hydrocarbons, compounds with toxic and carcinogenic properties. However, only a small number of hydroxylated polycyclic aromatic hydrocarbons have been determined in particles from wood combustion, usually compounds with available reference standards. In this present study, suspect and non-target screening strategies were applied to characterize the wood smoke particles from four different wood types and two combustion conditions with respect to hydroxylated polycyclic aromatic hydrocarbons and other organic compounds. In the suspect screening, 32 peaks corresponding to 12 monohydroxylated masses were tentatively identified by elemental composition assignments and matching of isotopic pattern and fragments. More than one structure was suggested for most of the measured masses. Statistical analysis was performed on the non-target screening data in order to single out significant peaks having intensities that depend on the wood type and/or combustion condition. Significant peaks were found in both negative and positive ionization modes, with unique peaks for each wood type and combustion condition, as well as a combination of both factors. Furthermore, structural elucidation of some peaks was done by comparing the spectra in the samples with spectra found in the spectral databases. Six compounds were tentatively identified in positive ionization mode, and 19 in negative ionization mode. The results in this present study demonstrate that there are significant overall differences in the chemistry of wood smoke particles that depends on both the wood type and the combustion condition used.

Determination of oxygenated and native polycyclic aromatic hydrocarbons in urban dust and diesel particulate matter standard reference materials using pressurized liquid extraction and LC-GC/MS.

The objective of this study was to develop a novel analytical chemistry method, comprised of a coupled high-performance liquid chromatography-gas chromatography/mass spectrometry system (LC-GC/MS) with low detection limits and high selectivity, for the identification and determination of oxygenated polycyclic aromatic hydrocarbons (OPAHs) and polycyclic aromatic hydrocarbons (PAHs) in urban air and diesel particulate matter. The linear range of the four OPAHs, which include 9,10-anthraquinone, 4H-cyclopenta[def]phenanthrene-4-one, benzanthrone, and 7,12-benz[a]anthraquinone, was 0.7 pg-43.3 ng with limits of detection (LODs) and limits of quantification (LOQs) on the order of 0.2-0.8 and 0.7-1.3 pg, respectively. The LODs in this study are generally lower than values reported in the literature, which can be explained by using large-volume injection. The recoveries of the OPAHs spiked onto glass fiber filters using two different pressurized liquid extraction (PLE) methods were in the ranges of 84-107 and 67-110 %, respectively. The analytical protocols were validated using the following National Institute of Standards and Technology standard reference materials: SRM 1649a (Urban Dust), SRM 1650b (Diesel Particulate Matter), and SRM 2975 (Diesel Particulate Matter, Industrial Forklift). The measured mass fractions of the OPAHs in the standard reference materials (SRMs) in this present study are higher than the values from the literature, except for benzanthrone in SRM 1649a (Urban Dust). In addition to the OPAHs, 44 PAHs could be detected and quantified from the same particulate extract used in this protocol. Using data from the literature and applying a two-sided t test at the 5 % level using Bonferroni correction, significant differences were found between the tested PLE methods for individual PAHs. However, the measured mass fractions of the PAHs were comparable, similar to, or higher than those previously reported in the literature.

A pragmatic definition of therapeutic synergy suitable for clinically relevant in vitro multicompound analyses.

For decades, the standard procedure when screening for candidate anticancer drug combinations has been to search for synergy, defined as any positive deviation from trivial cases like when the drugs are regarded as diluted versions of each other (Loewe additivity), independent actions (Bliss independence), or no interaction terms in a response surface model (no interaction). Here, we show that this kind of conventional synergy analysis may be completely misleading when the goal is to detect if there is a promising in vitro therapeutic window. Motivated by this result, and the fact that a drug combination offering a promising therapeutic window seldom is interesting if one of its constituent drugs can provide the same window alone, the largely overlooked concept of therapeutic synergy (TS) is reintroduced. In vitro TS is said to occur when the largest therapeutic window obtained by the best drug combination cannot be achieved by any single drug within the concentration range studied. Using this definition of TS, we introduce a procedure that enables its use in modern massively parallel experiments supported by a statistical omnibus test for TS designed to avoid the multiple testing problem. Finally, we suggest how one may perform TS analysis, via computational predictions of the reference cell responses, when only the target cell responses are available. In conclusion, the conventional error-prone search for promising drug combinations may be improved by replacing conventional (toxicology-rooted) synergy analysis with an analysis focused on (clinically motivated) TS.

Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics.

Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process.

Mandibular widening in hemifacial microsomia: a roentgen stereometric study of 11 patients with the aid of metallic implants.

INTRODUCTION: Our objective was to study mandibular widening in untreated subjects with hemifacial microsomia. METHODS: From the 3-dimensional files at the Department of Plastic and Reconstructive Surgery, Skane University Hospital in Malmö, Sweden, data of 11 subjects (3 girls, 8 boys) with hemifacial microsomia were retrieved. Their age range was 9 years 2 months to 13 years 2 months at the first examination. The mean observation period was 5 years 2 months. Each subject was studied by roentgen stereophotogrammetry with the aid of metallic implants. RESULTS: A significant widening of the mandible was found, with a mean total change of 0.31 mm (range, 0.08-0.79 mm) and a mean annual change of 0.07 mm (range, 0.03-0.12 mm). In 2 subjects, narrowing of the mandible was found: -0.16 and - 0.23 mm. CONCLUSIONS: The mandible got wider during adolescence in 9 of our subjects with hemifacial microsomia but to a lesser extent than has been reported in subjects without hemifacial microsomia and from ordinary orthodontic clinics. Sex difference was not addressed. It was suggested that chewing (forces and patterns) was responsible for the mandibular widening in our subjects; this is in line with previous research.

Dynamic hollow-fibre liquid phase microextraction of dinitrophenols from human plasma: optimization of an extraction flow system using experimental design methodology.

The utility of a dynamic hollow-fibre liquid phase microextraction method (optimized using a four-variable experimental design and response surface modelling) for extracting dinitrophenolic compounds from human plasma samples was evaluated. The investigated variables were donor phase salt concentration (10-400 mM), donor phase pH (2-6), acceptor phase pH (7-12), and donor/acceptor phase flow rates (30/7.5 to 70/17.5 microL min(-1)). Four dinitrophenol pesticides were used as model substances at concentrations of 0.1 microg mL(-1) in spiked human plasma samples. Extraction efficiencies ranging from 42 to 77% with RSDs below 9 were achieved with the optimized method. The flow rate and acceptor pH were shown to strongly affect the extraction efficiency for all compounds, while the donor phase pH and salt concentration had minor effects. With a well-defined acceptor phase pH and flow rate the system exhibited high robustness. The limits of quantification for the investigated compounds, using the presented extraction method followed by liquid chromatography/electrospray ionization mass spectrometry in selected ion monitoring mode, ranged from 0.05 to 0.1 microg mL(-1) plasma.

In vitro activity of 20 agents in different prognostic subgroups of chronic lymphocytic leukemia--rolipram and prednisolone active in cells from patients with poor prognosis.

BACKGROUND: There is a need for development of new drugs for treatment of B-cell chronic lymphocytic leukemia (CLL), especially for poor-prognostic subgroups resistant to conventional therapy. OBJECTIVE: The in vitro antileukemic activity of 20 different anticancer agents was characterized in tumor cells from CLL, aiming at identifying agents active in poor-prognostic subgroups. DESIGN AND METHODS: In tumor cells from 40 CLL patients and in peripheral blood mononuclear cells (PBMC) from three healthy controls, the activity of 20 substances was assessed using a non-clonogenic assay. The CLL samples were characterized regarding genomic aberrations by interphase fluorescence in situ hybridization and immunoglobulin heavy-chain variable (IGHV) gene mutational status. RESULTS: In line with clinical experience, cells from patients with unfavourable genomic aberrations [del(11q)/del(17p)] showed lower drug sensitivity to fludarabine and chlorambucil than cells from patients with favourable cytogenetics [del(13q)/no aberration]. Most investigated drugs demonstrated similar activity in CLL cells from patients with unmutated and mutated IGHV genes as well as in CLL cells vs. PBMC. Interestingly, prednisolone and rolipram displayed high CLL specificity, high activity in CLL cells with unmutated IGHV genes and retained the effect in several cases with 11q/17p deletion. Further studies on prednisolone and rolipram revealed a synergy when these agents were combined in CLL cells, and suggested correlation between drug sensitivity and difference in downstream signaling. CONCLUSION: Prednisolone and rolipram are interesting for further studies in CLL with inferior prognosis. The study can also be considered a basis for future efforts to find drugs active in subsets of CLL patients that are resistant to conventional therapy.

Rapamycin shows anticancer activity in primary chronic lymphocytic leukemia cells in vitro, as single agent and in drug combination.

The mammalian target of rapamycin inhibitor rapamycin and its analogues show promising anticancer activity in various experimental tumor models and are presently evaluated in clinical trials. We, here, evaluated the in vitro activity of rapamycin with regard to tumor-type specificity and possible mechanisms of drug resistance in 97 tumor cell samples from patients and in a resistance-based cell line panel, using the fluorometric microculture cytotoxicity assay. Rapamycin was dose-dependently cytotoxic in patient tumor cells and in cell lines. In primary cells, rapamycin was more active in hematological than in solid tumor samples, with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia being the most sensitive tumor types. Considerable inter-individual differences in sensitivity were apparent among CLL samples, but no difference was observed between IGHV mutated and unmutated CLL samples, whereas a tendency to lower rapamycin sensitivity was indicated for samples displaying poor-prognostic genomic markers. Combination experiments in CLL cells indicated that rapamycin acted synergistically with vincristine, cisplatin, chlorambucil and taxotere. These results and the clinically-experienced good tolerance to rapamycin analogues encourage clinical studies of rapamycin in CLL treatment as single agent but also in combination with, e.g., vincristine and chlorambucil.

Testicular prosthetic implants in boys and teenagers with primary or secondary anorchism.

From 1989 to 2005, 13 patients aged between 5 and 18 years had testicular prostheses implanted at the Department of Plastic and Reconstructive Surgery, University Hospital, Malmö, Sweden. The most common indication for surgery was aplastic or vanishing testes. In 13 patients and 19 operations 7 unilateral and 12 bilateral testicular prostheses were implanted. Each prosthesis was implanted through an inguinal skin incision, inserted digitally, and fixed with purse-string sutures. Some years after the primary implant three of the patients have had a second operation in which the small prosthesis was exchanged for an adult one. There was only one minor postoperative complication, a superficial wound infection. No patient required reoperation. All patients were happy with the results of surgery at the follow-up. We recommend this operation for implantation of testicular prostheses.

COX-2 and SCD, markers of inflammation and adipogenesis, are related to disease activity in Graves' ophthalmopathy.

CONTEXT: Inflammation and adipogenesis are two parallel processes with increased activity in severe Graves' ophthalmopathy. OBJECTIVE: The aim of this work was to define target genes for therapeutic intervention in adipogenesis and inflammation in Graves' ophthalmopathy. DESIGN: Orbital tissue was obtained from patients with ophthalmopathy in acute or chronic phase undergoing orbital surgery to study gene expression followed by the study of potential intervention mechanisms in preadipocytes. SETTING: Clinic of Endocrinology, University Hospital, Malmö, Sweden. PARTICIPANTS: Patients in acute severe or in chronic phase of ophthalmopathy. INTERVENTIONS: Lateral orbital decompression in acute phase and restorative surgery in chronic phase. In vitro treatment of preadipocytes with rosiglitazone and diclofenac. MAIN OUTCOME MEASURE: Gene expression in intraorbital tissue or preadipocytes and differentiation of preadipocytes. RESULTS: A marker of adipose tissue, stearoyl-coenzyme A desaturase (SCD), and the proinflammatory gene, cyclooxygenase-2 (COX-2), were overexpressed in patients in active phase compared to the chronic phase of ophthalmopathy. In growth-arrested preadipocytes stimulated with rosiglitazone, COX-2 expression increased temporarily within 1 hour and decreased to undetectable levels after 48 hours. In contrast, SCD and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression increased continuously from day 2 to day 7 during adipogenesis. Diclofenac, an inhibitor of cyclooxygenases with antagonistic effects on PPAR-gamma, reduced the number of mature adipocytes by approximately 50%. CONCLUSION: We conclude that inflammation and adipogenesis decrease with a decrease in activity of ophthalmopathy and that the nonsteroidal antiinflammatory drug diclofenac inhibits adipogenesis. This may represent a putative future treatment of endocrine ophthalmopathy.

Analysis of complications after repair of hypospadias.

We have reviewed 184 patients after repair of primary hypospadias. They were operated on and followed-up according to a standard protocol, and the senior author (HS) participated in all operations. The patients were reconstructed using a Byars two-stage procedure (n=102) or a Scuderi (n=29) or Mathieu (n=41) single-stage procedure. The rest (n=12) just had a removal of the chordee with realignment of the skin. Complications recorded were haematomas, postoperative infections, malfunction of catheter, urinary retention, fistulas, and strictures. Thirty-eight complications developed in 26 patients and the overall incidence of fistula was 22 in 18 patients (10%). Our findings suggest that the most decisive risk factor for complications is the severity of the primary malformation, because a severe malformation per se is difficult to treat as it requires a long reconstruction; in addition the curvature, shortage of tissue, and extensive surgery generally require a staged reconstruction in these cases. Other factors seem to be of much lesser importance.

Thyrostimulin (a TSH-like Hormone) expression in orbital and thyroid tissue.

OBJECTIVE: To investigate gene expression of thyrostimulin in orbital and thyroid tissue from patients with and without Graves' disease. DESIGN: Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used for detection of thyrostimulin gene expression in intraorbital adipose tissue from patients with severe ophthalmopathy and thyroid healthy controls in addition to thyrostimulin expression in normal thyroid tissue, multinodular goiter tissue, and Graves' thyroid tissue. MAIN OUTCOME: In intraorbital tissue, thyrostimulin expression was identified in both patients and controls with fluorescence intensities varying between 0.23 and 0.88 in patients and 0.29 and 8.9 in controls before treatment with DNase. The signal of thyrostimulin was weak or absent in intraorbital adipose tissue from patients with ophthalmopathy and thyroid healthy controls after treatment of samples with DNase. This was in contrast to the expression of the thyroid-stimulating hormone (TSH) receptor and the housekeeping gene cyclophilin A that were detected both before and after DNase treatment. Similar results were found when analyzing human and rat thyroid tissue. CONCLUSIONS: Neither did we demonstrate gene expression of thyrostimulin in intraorbital adipose tissue or in thyroid tissue, nor could we confirm earlier findings in rat thyroid tissue. Whether thyrostimulin is a regulator of thyroid function has to be further investigated in future studies.

Overexpression of immediate early genes in active Graves' ophthalmopathy.

CONTEXT: In Graves' ophthalmopathy a major problem is an increase in the intraorbital adipose tissue volume. OBJECTIVE: The aim of this work was to define mechanisms of orbital adipogenesis. DESIGN: This was an open-label prospective study. SETTING: The study was conducted at the Clinic of Endocrinology, University Hospital. PARTICIPANTS: The study consisted of patients (n = 5) with severe ophthalmopathy with affection of the optic nerve and thyroid healthy controls (n = 5). INTERVENTIONS: We performed lateral decompression of orbital tissue in patients unresponsive to corticosteroids and restorative surgery of the upper eyelid in thyroid healthy controls. MAIN OUTCOME MEASURE: We made large-scale measurements of gene expression, with microarray technique based on determination of fluorescence intensities in cases and controls. RESULTS: A marker of adipose tissue, stearoyl-coenzyme A desaturase, was overexpressed in ophthalmopathy, and selection criteria were set to favor identification of genes known to be expressed in normal adipogenesis. The immediate early gene, cysteine-rich, angiogenic inducer, 61 (CYR61), was overexpressed in addition to 15 other immediate early genes (IEGs), and the expression of selected IEGs was confirmed with RT-PCR: CYR61, cyclooxygenase-2, dual-specificity phosphatase 1, B cell translocation gene 2, and early growth response 1. CYR61-responsive genes, known to participate in inflammation, IL-1beta, matrix metalloproteinase-3, and vascular endothelial growth factor were also overexpressed. Patients showed greater expression of CYR61 in the active than the chronic phase of ophthalmopathy, indicating that CYR61 is a marker of disease activity. Cyclooxygenase-2, the target gene of IL-1beta, was also overexpressed, although all patients had been treated with corticosteroids. CONCLUSION: Adipocyte-related IEGs are overexpressed in active ophthalmopathy, and CYR61 may have a role in both orbital inflammation and adipogenesis and serve as a marker of disease activity.

Split skin graft reconstruction in vaginal agenesis: a long-term follow-up.

Fifteen women who had isolated congenital vaginal agenesis reconstructed by the split skin inlay grafting technique at a mean age of 19 (0.5) years, were followed up 2-33 years after the operation. The reconstruction had been functionally successful in all patients. No patient reported dyspareunia, exceptional dryness, or disturbing vaginal discharge. All the women had established normal, stable relationships with men. Their general recollection of the hospital stay and treatment was favourable. The importance of a thorough preoperative and postoperative explanation was emphasised by the patients, as well as their wish for long term contact with a specialist in gynaecology who was well aware of their special problems. In conclusion, the good functional results have been accompanied by stable social and psychological relationships.