Two types of autologous cells in stricture development prevention after complete circular endoscopic dissection in minipig.

INTRODUCTION: Complete circular endoscopic dissection (CED) is frequently accompanied with post-operative strictures formation in the esophagus. Various types of therapeutic approaches have recently been tested to prevent these strictures, e.g. cell therapy or stenting. METHODS: Miniature pigs of Gottingen/Minnesota origin (n=10) were used in the study. First, we made the complete CED in the mid esophagus; next, the defect was left untreated or covered with mesenchymal stem cells (MSCs) or a mixture of MSCs and primary oral keratinocytes (pOKs) suspension without/with fully covered self-expandable metallic stent (SEMS). Consequently, we performed a control endoscopy with a stent removal, and necropsy was performed 17-36 days after cells application. RESULTS: All CED procedures were completed successfully without serious complications. Although we were able to detect MSCs or pOKs in the post-CED defects up to the 36th day after transplantation, the combination of MSCs or MSCs/pOKs with or without SEMS application did not prevent post-CED strictures development. The mixture of MSCs and pOKs resulted in the formation of cellular aggregates, which were mainly observed in submucosa, and the post-CED defect was covered with collagen fibers containing a thin scarred epithelium, accompanied by various degrees of reconstruction and integrity. CONCLUSION: Suspension application of autologous MSCs alone or in combination with pOKs with or without SEMS was ineffective in the prevention of strictures formation after complete CED. Nevertheless, the presence of MSCs or pOKs in the post-CED defect was confirmed even 5 weeks after transplantation.

Iron deficiency and fatigue in adolescent females with heavy menstrual bleeding.

Iron deficiency and fatigue are common problems in adolescent females. Heavy menstrual bleeding (HMB) is associated with both iron deficiency and fatigue. The aim of this study was to define baseline ferritin values and fatigue symptoms in a population of young females with excessive menstrual blood loss, as compared to healthy controls. The study population included 11 to 17-year-old menstruating females presenting to an Adolescent Gynaecology Clinic, Menorrhagia Clinic or Sports Medicine clinic. To evaluate the degree and effects of menstrual blood loss, we utilized the Ruta Menorrhagia Severity Score. We investigated the symptoms of fatigue using the Fatigue Severity Scale. We evaluated possible predictors of ferritin level (age, body mass index, fatigue scores and Menorrhagia Severity Score) using generalized linear models. A total of 48 adolescents with HMB and 102 healthy adolescents completed the study. Iron deficiency and elevated fatigue scores were common findings in young women with HMB. Both fatigue severity scores and menorrhagia severity scores were significantly higher in young women with HMB as compared to healthy controls. In adolescents with HMB, 87.5% had ferritin levels ≤40 ng mL(-1), and 29.2% had ferritin levels ≤15 ng mL(-1). Our generalized linear models did not identify any significant univariate relationships between ferritin levels and patient age, body mass index, fatigue score or menorrhagia score. Iron deficiency and symptoms of fatigue are common findings in young women with HMB. Fatigue severity scores are significantly higher in young women with HMB as compared to healthy controls.

Menstrual bleeding patterns and prevalence of bleeding disorders in a multidisciplinary adolescent haematology clinic.

Heavy menstrual bleeding (HMB) is a frequent complaint in adolescence. Although HMB is often caused by immaturity of the hypothalamic-pituitary-ovarian axis, bleeding disorders are another common yet often unidentified cause. The aim of this study was to examine the bleeding patterns and prevalence of inherited bleeding disorders among females referred for HMB to a multidisciplinary adolescent haematology clinic. We retrospectively reviewed the first 105 patients (ages 8-18 years) referred to this specialty clinic from February 2009 to December 2011. Using menstrual bleeding questionnaires and medical records, data were extracted regarding demographics, bleeding patterns, frequency and types of bleeding disorders identified, and prescribed interventions. Sixty-two per cent of patients were diagnosed with a bleeding disorder, including platelet storage pool deficiency (36%), von Willebrand's disease (9%), other platelet function defect (8%), Ehlers-Danlos syndrome (7%) and combined bleeding disorders (2%). Comparison of the bleeding profiles for females with and without a bleeding disorder revealed only three factors that were significantly different, including the reported regularity of patients' periods (P = 0.02), description of period flow (P = 0.04) and number of days of each period that the bleeding was described as 'heavy' (P = 0.007). Bleeding disorders are prevalent in adolescent females presenting to a specialty clinic. Specifically, a relatively high proportion of adolescents were diagnosed with platelet storage pool deficiency. In our small population, menstrual bleeding profiles, as examined by a standardized questionnaire, could not identify females with an underlying bleeding disorder, demonstrating the important role of haemostasis testing in the evaluation of adolescents with HMB.

The PFA-100 ® does not predict delta-granule platelet storage pool deficiencies.

There are no published reports investigating the ability of the platelet function analyzer (PFA-100(®) ) to detect the presence of delta-granule platelet storage pool deficiencies (δ-PSPD), a common mild bleeding disorder. Prior studies of the PFA-100(®) and congenital platelet disorders have been limited by small numbers of patients with a variety of disorders. We examined PFA-100(®) results in a large paediatric patient population diagnosed specifically with δ-PSPD, and determined the relationship between PFA-100(®) and platelet electron microscopy (the gold standard for diagnosis). This study is a retrospective review of patients <19 years of age diagnosed with δ-PSPD at Nationwide Children's Hospital from 2008 to 2010. To examine the correlation between PFA-100(®) and average number of granules per platelet we used Spearman's Rho as a non-parametric measure of dependence. A total of 105 patients diagnosed with δ-PSPD were included, of which 99 patients underwent PFA-100(®) testing. Of those tested 46% had at least one abnormal closure time, whereas 16% had abnormal results for both cartridges. We found no statistical correlation between C-EPI closure time and average number of granules per platelet (ρ= -0.0095, P-value = 0.9328), nor between C-ADP closure time and the average number of granules (ρ = 0.0315, P-value = 0.7798). The PFA-100(®), a widely used screening test for suspected bleeding disorders, did not correlate with presence or severity of δ-PSPD as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA-100(®) alone cannot be used to rule out the presence of a δ-PSPD.

Transduction profiles in minipig following MRI guided delivery of AAV-5 into thalamic and corona radiata areas.

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

The power of a standardized bleeding score in diagnosing paediatric type 1 von Willebrand's disease and platelet function defects.

A predictive standardized bleeding questionnaire (Vicenza score), previously validated for identifying individuals with type 1 von Willebrand's disease (VWD), has never been prospectively validated in tertiary care paediatric settings. The aim of this study was to assess the Vicenza score's predictive power in identifying type 1 VWD, low von Willebrand factor (VWF) and platelet function defects (PFD) in a prospective cohort of patients, 0-17 years old, referred to a paediatric haematology clinic for evaluation of a bleeding disorder. Before the initial visit, caregivers consented to answer the questionnaire via telephone. Patients' medical records were reviewed after haematological evaluation. VWF:Ag or VWF:RCo<30 IU dL(-1) were labelled 'definite type 1 VWD' while 30-50 IU dL(-1) were labelled 'Low VWF'. PFA-100 screening followed by abnormal electron microscopy and/or platelet aggregation studies diagnosed a PFD. At least one haemorrhagic symptom was present in 99 of the 104 children who completed the study (mean number of symptoms 2.87, mean Vicenza score 3.24). Eight met criteria for 'definite type 1 VWD', 23 for 'low VWF' and 13 for 'PFD'. The sensitivity, specificity, and positive and negative predictive value (NPV) of the Vicenza score demonstrated poor diagnostic utility with the exception of high specificity in ruling out 'definite type 1 VWD'. The NPV was comparably high with qualitative (>2 bleeding symptoms) and quantitative (Vicenza score ≥ 2) criteria. The Vicenza score has limited predictive value in paediatric tertiary care settings. While the NPV of excluding 'definite type 1 VWD' is high, simpler qualitative criteria is similarly predictive.

Quality of newly formed cartilaginous tissue in defects of articular surface after transplantation of mesenchymal stem cells in a composite scaffold based on collagen I with chitosan micro- and nanofibres.

The aim of this study was to evaluate macroscopically, histologically and immunohistochemically the quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle in miniature pigs treated with the clinically used method of microfractures in comparison with the transplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells (MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects of articular cartilage after transplantation of the scaffold with MSCs (Group A) had in 60 % of cases a macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone (Group B) or after the method of microfractures (Group C), erosions/fissures or osteophytes were found on the surface. The results of histological and immunohistochemical examination using the modified scoring system according to O'Driscoll were as follows: 14.7+/-3.82 points after transplantations of the scaffold with MSCs (Group A); 5.3+/-2.88 points after transplantations of the scaffold alone (Group B); and 5.2+/-0.64 points after treatment with microfractures (Group C). The O'Driscoll score in animals of Group A was significantly higher than in animals of Group B or Group C (p<0.0005 both). No significant difference was found in the O'Driscoll score between Groups B and C. The treatment of iatrogenic lesions of the articular cartilage surface on the condyles of femur in miniature pigs using transplantation of MSCs in the composite scaffold led to the filling of defects by a tissue of the appearance of hyaline cartilage. Lesions treated by implantation of the scaffold alone or by the method of microfractures were filled with fibrous cartilage with worse macroscopic, histological and immunohistochemical indicators.

Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing.

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

Regionally-selective cell colonization of micropatterned surfaces prepared by plasma polymerization of acrylic acid and 1,7-octadiene.

Micropatterned surfaces have been used as a tool for controlling the extent and strength of cell adhesion, the direction of cell growth and the spatial distribution of cells. In this study, chemically micropatterned surfaces were prepared by successive plasma polymerization of acrylic acid (AA) and 1,7-octadiene (OD) through a mask. Rat vascular smooth muscle cells (VSMC), bovine endothelial cells (EC), porcine mesenchymal stem cells (MSC) or human skeletal muscle cells (HSKMC) were seeded on these surfaces in densities from 9,320 cells/cm(2) to 31,060 cells/cm(2). All cell types adhered and grew preferentially on the strip-like AA domains. Between day 1 and 7 after seeding, the percentage of cells on AA domains ranged from 84.5 to 63.3 % for VSMC, 85.3 to 73.5 % for EC, 98.0 to 90.0 % for MSC, and 93.6 to 55.0 % for HSKMC. The enzyme-linked immunosorbent assay (ELISA) revealed that the concentration of alpha-actin per mg of protein was significantly higher in VSMC on AA. Similarly, immunofluorescence staining of von Willebrand factor showed more apparent Weibel-Palade bodies in EC on AA domains. MSC growing on AA had better developed beta-actin cytoskeleton, although they were less stained for hyaluronan receptor (CD44). In accordance with this, MSC on AA contained a higher concentration of beta-actin, although the concentration of CD44 was lower. HSKMC growing on AA had a better developed alpha-actin cytoskeleton. These results based on four cell types suggest that plasma polymerization is a suitable method for producing spatially defined patterned surfaces for controlled cell adhesion, proliferation and maturation.

Differential regulation of galectin expression/reactivity during wound healing in porcine skin and in cultures of epidermal cells with functional impact on migration.

The glycophenotyping of mammalian cells with plant lectins maps aspects of the glycomic profile and disease-associated alterations. A salient step toward delineating their functional dimension is the detection of endogenous lectins. They can translate sugar-encoded changes into cellular responses. Among them, the members of the lectin family of galectins are emerging regulators of cell adhesion, migration and proliferation. Focusing on galectins-1, -3 and -7, we addressed the issue whether their expression is regulated during wound healing in porcine skin as model. A conspicuous upregulation is detected for galectin-1 in the dermis and a neoexpression in the epidermis, where an increased level of galectin-7 was also found. Applying biotinylated tissue lectins as probes, the signal intensities for accessible binding sites decreased, intimating an interaction of the cell lectin with reactive sites. In contrast, galectin-3 parameters remained rather constant. Of note, epidermal cells in culture also showed an increase in expression/presence of galectin-1, measured on the levels of mRNA and protein, in this case by Western blotting and quantitative immunocytochemistry. Used as matrix, galectin-1 conferred resistance to trypsin treatment to attached human keratinocytes and reduced migration into scratch-wound areas in vitro. This report thus presents new information on endogenous lectins in wound healing and differential regulation among the three tested cases.

FEM simulation of a sono-reactor accounting for vibrations of the boundaries.

The chemical effects of acoustic cavitation are obtained in sono-reactors built-up from a vessel and an ultrasonic source. In this paper, simulations of an existing sono-reactor are carried out, using a linear acoustics model, accounting for the vibrations of the solid walls. The available frequency range of the generator (19-21 kHz) is systematically scanned. Global quantities are plotted as a function of frequency in order to obtain response curves, exhibiting several resonance peaks. In absence of the precise knowledge of the bubbles size distribution and spatial location, the attenuation coefficient of the wave is taken as a variable, but spatially uniform parameter, and its influence is studied. The concepts of acoustic energy, intensity, active power, and source impedance are recalled, along with the general balance equation for acoustic energy, which is used as a convergence check of the simulations. It is shown that the interface between the liquid and the solid walls cannot be correctly represented by the simple approximations of either infinitely soft, or infinitely hard boundaries. Moreover, the liquid-solid coupling allows the cooling jacket to receive a noticeable part of the input power, although it is not in direct contact with the sonotrode. It may therefore undergo cavitation and this feature opens the perspective to design sono-reactors which avoid direct contact between the working liquid and the sonotrode. Besides, the possibility to shift the main pressure antinode far from the sonotrode area by exciting a resonance of the system is examined.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study.

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.

Rapid determination of omeprazole in human plasma by protein precipitation and liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and reliable method was developed to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The assay is based on protein precipitation with acetonitrile and reversed-phase liquid chromatography performed on an octadecylsilica column (55 mm x 2mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (60:40, v/v). Omeprazole and flunitrazepam, the internal standard, elute at 0.80+/-0.10 min with a total run time 1.35 min. Quantification was through positive ion mode and selected reaction monitoring mode at m/z 346.1-->197.9 for omeprazole and m/z 314.0-->268.0 for flunitrazepam, respectively. The lower limit of quantitation was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 12%. The assay was applied to the analysis of samples from a pharmacokinetic study.

Energetic balance in an ultrasonic reactor using focused or flat high frequency transducers.

In order to undertake irradiation of polymer blocks or films by ultrasound, this paper deals with the measurements of ultrasonic power and its distribution within the cell by several methods. The electric power measured at the transducer input is compared to the ultrasonic power input to the cell evaluated by calorimetry and radiation force measurement for different generator settings. Results obtained in the specific case of new transducer types (composites and focused composites i.e., HIFU: high intensity focused ultrasound) provide an opportunity to conduct a discussion about measurement methods. It has thus been confirmed that these measurement techniques can be applied to HIFU transducers. For all cases, results underlined the fact that measurement of radiation pressure for power evaluation is more adapted to low powers (<15 W) and that measurement by calorimetry is a valid technique for global energy measurements. Composites and monocomponent transducers were compared and it appears that the presence of an adaptation glass plate reduces the efficiency of the monocomponent transducers. The distribution of ultrasonic intensity is qualitatively depicted by sono-chemiluminescence of luminol. Finally, the quantity of energy absorbed by samples placed in the sound field is determined and the temperature distribution monitored as a function of wall distance. This energetic balance allows us to understand the global behaviour of all experimental set-ups made up of a generator-transducer-liquid and sample.

Optimisation of 20 kHz sonoreactor geometry on the basis of numerical simulation of local ultrasonic intensity and qualitative comparison with experimental results.

The intensity distribution of the ultrasonic energy is, after the frequency, the most significant parameter to characterize ultrasonic fields in any sonochemical experiment. Whereas in the case of low intensity ultrasound the measurement of intensity and its distribution is well solved, in the case of high intensity (when cavitation takes place) the measurement is much more complicated. That is why the predicting the acoustic pressure distribution within the cell is desirable. A numerical solution of the wave equation gave the distribution of intensity within the cell. The calculations together with experimental verification have shown that the whole reactor behaves like a resonator and the energy distribution depends strongly on its shape. The agreement between computational simulations and experiments allowed optimisation of the shape of the sonochemical reactor. The optimal geometry resulted in a strong increase in intensity along a large part of the cell. The advantages of such optimised geometry are (i) the ultrasonic power necessary for obtaining cavitation is low; (ii) low power delivered to the system results in only weak heating, consequently, no cooling is necessary and (iii) the "active volume" is large, i.e. the fraction of the reactor volume with high intensity is large and is not limited to a vicinity close to the horn tip.

Porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation.

This article summarizes research using cells derived from epidermis of the miniature pigs for use as a cell therapy for skin repair and as a model for squamous carcinoma of the head and neck. Stem cells are an important "tool" for biomedical research. Adult stem cells are defined functionally, as cells that have the capacity to self-renew as well as the ability to generate differentiated cells. They are present in defined tissue microenvironments called niches. Asymmetric mitosis allows them to produce one daughter cell with the properties of stem cells (self-renewal) and a second cell with characteristics of progenitor cells, or transit amplifying cells, which proliferate quickly but with a limited number of mitotic divisions. Porcine epidermal stem cells, located in the bulge region of the outer root sheath of hair follicles, migrate in vitro from hair sheaths and because they are resistant to anoikis (detachment induced apoptosis), survive in non-adhesive conditions to form spheroids. These cells express keratins, galectin-1 and their nuclei are rich in DeltaNp63alpha. Interestingly, the multiple phenotype analysis of the human tumor cells in squamous carcinoma of head and neck revealed similarities with epidermal stem cells. These cancer stem cells are usually located on the periphery of the tumor where the invasive front of the tumor responsible for its aggressive behavior is located. In contrast, extensive expression of markers of terminal differentiation such as expression of glycoligands reactive for the endogenous lectin, galectin-3, indicates better tumor prognosis.

Phenotypic characterization of porcine interfollicular keratinocytes separated by elutriation: a technical note.

Separation of epidermal stem cells from other populations in suspensions of epidermal cells by sorting is hampered by a present lack of specific surface markers of this cell type. To address this issue we applied CCE combined with immunocytochemical phenotyping. On the basis of expression profiles for keratins (10, 14, and 19), nucleostemin, galectin-1 and epitopes reactive for this adhesion/growth-regulatory tissue lectin we identified a fraction of very small cells originating from the basal layer. The results demonstrated that CCE has potential merit for separation of epidermal cells to yield a population likely enriched in stem cells.

Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm x 4 mm, 5 microm particles), the mobile phase consisted of acetonitrile -15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.

Preparation and characterization of poly-(D,L-lactide-co-glycolide) and poly-(L-lactic acid) microspheres with entrapped pneumotropic bacterial antigens.

Poly-(lactide-co-glycolide) microspheres with entrapped antigen have shown considerable promise as controlled release vaccines. To enhance the immunomodulatory effect of LW 50020, a bacterial lysate of seven common respiratory pathogens used perorally as an immunomodulator, we prepared poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres with entrapped immunomodulator by solvent evaporation or solvent extraction double emulsion techniques. Physical properties, such as particle size, LW 50020 entrapment rate, antigen release patterns and morphological characteristics were investigated. All preparations displayed a high degree of antigen loading up to 95%, whereas size, surface morphology and antigen release patterns were significantly influenced by the method of preparation and the polymer components used. Solvent evaporation microspheres are porous particles from 0.8 micron to 2.0 microns in diameter, that show a rapid antigen release for PLG, and a moderate antigen release for PLA microspheres within 33 days. Solvent extraction microspheres have proven to be particles from 1.1 microns to 5.0 microns in diameter showing a smooth surface and a medium antigen release rate over 33 days. SDS-PAGE and immunoblotting of extracted antigen confirmed that the molecular weight and antigenicity of the immunomodulator remained unaltered by the entrapment procedure.

Intra-thymic nurse cell lymphocytes can induce a graft-versus-host reaction with high efficiency.

Thymic nurse cells (TNC) are multicellular complexes composed of morphologically intact thymocytes internalized within epithelial cells. Due to their unusual features, they have been suspected to provide an appropriate microenvironment for T-cell repertoire acquisition. In recent functional studies in the chicken system, we have shown that intra-thymic nurse cell-lymphocytes (TNC-L) are highly efficient inducers of a graft-versus-host reaction (GvHR) in an allogeneic chorionallantoic membrane (CAM) assay, manifested by the formation of visible colonies, so-called pocks. Immunohistochemical and electron microscopical assessment of pocks revealed no differences between the cellular components and ultrastructural features of GvHR induced by TNC-L or peripheral blood lymphocytes (PBL). In addition, the stimulation of this local GvHR by interleukin 2 (IL-2) was demonstrated.