Additional assessment of Acute Cystitis Symptom Score questionnaire for patient-reported outcome measure in female patients with acute uncomplicated cystitis: part II.

PURPOSE: Since symptomatic, non-antibiotic therapy has become an alternative approach to treat acute cystitis (AC) in women, suitable patient-reported outcome measures (PROM) are urgently needed. The aim of this part II of a larger non-interventional, case-control study was the additional assessment of the ACSS as a suitable PROM. METHODS: Data from 134 female patients with diagnosed acute uncomplicated cystitis were included in the current analysis with (1) a summary score of "Typical" domain of 6 and more; (2) at least one follow-up evaluation after the baseline visit; (3) no missing values in the ACSS questionnaire data. Six different predefined thresholds based on the scoring of the ACSS items were evaluated to define "clinical cure", also considering the draft FDA and EMA guidelines. RESULTS: Of the six different thresholds tested, a summary score of the five typical symptoms of 5 and lower with no symptom more than 1 (mild), without visible blood in urine, with or without including QoL issues was favoured, which partially also could be adapted to the draft FDA and EMA guidelines. The overall patient's clinical assessment ("Dynamic" domain) alone was not sensitive enough for a suitable PROM. CONCLUSIONS: Scoring of the severity of symptoms is needed not only for diagnosis, but also for PROM to define "clinical cure" of any intervention, which could be combined with QoL issues. Results of the study demonstrated that the ACSS questionnaire has the potential to be used as a suitable PROM and should further be tested in prospective clinical studies.

Identification of the gene product recognized by monoclonal antibody GIIF3.

The chicken as a research model has a disadvantage compared with the mouse and the human because of the low number of available antibodies against gene products of interest. The goal of this study was to identify the antigen recognized by monoclonal antibody (mAb) GIIF3, which is a 42 kDa protein that appears in follicle-associated epithelium of the guinea hen as well as in different muscle types during chicken embryonic development. The 42 kDa protein, immunoprecipitated from chicken gizzard protein lysates, was evaluated by mass spectrometry. Mass spectrometry analysis revealed peptides specific for the chicken ß- or γ-actin isoforms. The mAb GIIF3 can be used as a new research tool for smooth muscle cell and bursa of Fabricius developmental studies.

Durable improvements in urinary incontinence and positive treatment response in patients with idiopathic overactive bladder syndrome following long-term onabotulinumtoxinA treatment: Final results of 3.5-year study.

OBJECTIVES: Here we present the final results from an extension study assessing long-term onabotulinumtoxinA treatment (3.5 years) in patients with idiopathic overactive bladder. METHODS: Patients who completed either of 2 Phase III trials were eligible to enter a 3-year extension study in which they received multiple onabotulinumtoxinA (100 U) treatments. Data were analyzed for the overall population of patients who received 100 U in any treatment cycle (n=829) and within discrete subgroups of patients who received exactly 1 (n=105), 2 (n=118), 3 (n=117), 4 (n=83), 5 (n=46), or 6 (n=33) treatments of the 100 U dose throughout the study (n=502). RESULTS: Of the 829 patients enrolled, 51.7 % completed the study. Discontinuations due to AEs/lack of efficacy were low (5.1/5.7 %); other reasons were not treatment-related. Mean reductions from baseline in urinary incontinence (UI) episodes/day (week 12; co-primary endpoint) were consistent among discrete subgroups who received 1 (-3.1), 2 (-2.9, -3.2), 3 (-4.1 to -4.5), 4 (-3.4 to -3.8), 5 (-3.0 to -3.6), or 6 (-3.1 to -4.1) treatments. A consistently high proportion of patients reported improvement/great improvement on the Treatment Benefit Scale (week 12; co-primary endpoint) in the discrete subgroups across all treatments (70.0-93.5 %). Median time to request retreatment was ≤6 months for 34.2 %, >6-≤12 months for 37.2 %, and >12 months for 28.5 % of patients. Most common AE was UTI, with no changes in safety profile over time. CONCLUSION: Long-term onabotulinumtoxinA treatment resulted in consistent reductions in UI and high proportions of patients reporting improvement after each treatment, with no new safety findings.

New approaches to nanoparticle sample fabrication for atom probe tomography.

Due to their unique properties, nano-sized materials such as nanoparticles and nanowires are receiving considerable attention. However, little data is available about their chemical makeup at the atomic scale, especially in three dimensions (3D). Atom probe tomography is able to answer many important questions about these materials if the challenge of producing a suitable sample can be overcome. In order to achieve this, the nanomaterial needs to be positioned within the end of a tip and fixed there so the sample possesses sufficient structural integrity for analysis. Here we provide a detailed description of various techniques that have been used to position nanoparticles on substrates for atom probe analysis. In some of the approaches, this is combined with deposition techniques to incorporate the particles into a solid matrix, and focused ion beam processing is then used to fabricate atom probe samples from this composite. Using these approaches, data has been achieved from 10-20 nm core-shell nanoparticles that were extracted directly from suspension (i.e. with no chemical modification) with a resolution of better than ± 1 nm.

Geochemical recovery of the Torna-Marcal river system after the Ajka red mud spill, Hungary.

The failure of the Ajka red mud depository in October 2010 led to the largest single release of red mud into the surface water environment. This study provides a comparative assessment of stream sediment quality in the Torna-Marcal-Rába catchment between post-disaster surveys (2010) and follow up surveys at an identical suite of 21 locations in 2013. The signature of red mud apparent in initial surveys with high Al, As, Cr, Na, V was only apparent at a small number of sample stations in recent surveys. These constitute <1 km of stream, compared to the >20 km reach of affected sediments in the immediate aftermath of the spill. Concentrations of red mud-derived contaminants are predominately associated with fine fractions of the red mud (<8 µm). This enhances transport out of the system of red mud-derived contaminants and, along with extensive remedial efforts, has substantially limited the within-channel inventory of potentially ecotoxic metals and metalloids.

The fibrin-derived citrullinated peptide ß60-74Cit60,72,74 bears the major ACPA epitope recognised by the rheumatoid arthritis-specific anticitrullinated fibrinogen autoantibodies and anti-CCP2 antibodies.

OBJECTIVES: To evaluate the proportions of rheumatoid arthritis (RA) sera containing anticitrullinated proteins autoantibodies (ACPA) reactive to α36-50Cit38,42 and/or ß60-74Cit60,72,74, two peptides identified as bearing the immunodominant epitopes of their major target, citrullinated fibrin. To analyse the relationships of anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies with autoantibodies reactive to the complete citrullinated human fibrinogen molecule (AhFibA) and with anti-CCP2 antibodies. METHODS: 617 sera from 181 patients with established RA and 436 with non-RA rheumatic diseases were tested by ELISA for AhFibA, anti-CCP2, anti-α36-50Cit38,42, anti-ß60-74Cit60,72,74 autoantibodies, and by nephelometry for rheumatoid factor (RF). Diagnostic indexes, correlations and concordances between tests were analysed. Crossreactivity of anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies was assessed in competition experiments. RESULTS: At a diagnostic specificity of 95%, the diagnostic sensitivity of AhFibA (83%) was significantly higher than that of all other tests. The diagnostic sensitivity of anti-ß60-74Cit60,72,74 (71%) was significantly higher than that of anti-α36-50Cit38,42 autoantibodies (51%) but similar to that of anti-CCP2 (74%). Titres of RF, anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 autoantibodies were weakly correlated with each other, whereas titres of anti-ß60-74Cit60,72,74 were strongly correlated with those of AhFibA (r=0.633) and anti-CCP2 (r=0.634). Anti-α36-50Cit38,42 and anti-ß60-74Cit60,72,74 mainly corresponded to two non-crossreactive subfamilies of ACPA. More than 90% of AhFibA-positive or anti-CCP2-positive sera recognised the α36-50Cit38,42 and/or the ß60-74Cit60,72,74 peptide. CONCLUSIONS: Autoantibodies reactive to α36-50Cit38,42 and ß60-74Cit60,72,74 form two distinct, non-overlapping subfamilies of ACPA that, together, cover practically all the ACPA reactivity to citrullinated fibrinogen and to CCP2 antigens. In established RA, anti-ß60-74Cit60,72,74 autoantibodies show diagnostic indexes similar to those of anti-CCP2.

Phylogenetic diversity and functional efficacy of the C-terminally expressed heptapeptide unit in the opioid precursor polypeptide proenkephalin A.

The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met- and Leu-enkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identified among 15 animals, such as YGGFMGY (zebrafish), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel heptapeptide, together with the mammalian consensus MERF and Met-enkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes. Equilibrium binding affinities changed from good to modest as measured by receptor type selective [3H]opioid radioligands. The relative affinities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [35S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green fluorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution.

Bioinformatic and biochemical studies on the phylogenetic variability of proenkephalin-derived octapeptides.

Leu- and Met-enkephalins are proenkephalin-derived endogenous pentapeptides with opioid (morphine) activity. Among the seven enkephalin units found in the human proenkephalin (PENK), the fourth copy being an octapeptide: Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu ((Hs)YGGFMRGL). Bioinformatic analysis of the available PENK sequences revealed the presence of other octapeptide orthologues in these precursor polypeptides. Four types of the elongated Met-enkephalins we identified by searching protein databases, (Xl)YGGFMRGY (three frog species and platypus), (Gg)YGGFMRSV (chicken and one fish species), (Hp)YGGFMNGF (shark) and (Mm)YGGFMRSL (mouse and two lungfish species) were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. All peptides have also been prepared containing oxidized methionine (M(O)). The overall binding and signalling profile of the novel octapeptides revealed moderate opioid agonist activities and a rank order of potencies for the mu approximately delta>>kappa receptor binding sites. Peptides with the oxidized M(O) residue were found to be less potent in both receptor binding and G-protein stimulation studies. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library approach is expected.

Binding studies of novel, non-mammalian enkephalins, structures predicted from frog and lungfish brain cDNA sequences.

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the kappa-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [(35)S]GTPgammaS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.

Identification of the avian B-cell-specific Bu-1 alloantigen by a novel monoclonal antibody.

A panel of monoclonal antibodies was generated against the guinea fowl's bursal cells. One of the antibodies, designated BoA1, recognized both cortical and medullary B cells of bursal follicles and B cell dependent regions of peripheral lymphoid organs, like germinal centers and splenic periellipsoidal regions. The staining pattern of this monoclonal antibody is similar to other antibodies (L22, 11G2, AV20), which also identify the Bu-1 antigens. Under reducing conditions, the molecular weight of the BoA1 antigen is 70 to 73 kDa, and after immunoprecipitation it proved to be identical with the antigen recognized by the AV20 antibody. It is unique for this novel monoclonal antibody that it shows wide range cross-reactivity with different avian species, like chicken, quail, guinea fowl, and turkey. Therefore, this Bu-1-specific monoclonal antibody could be a versatile tool for studying the B cell development in different domesticated birds.

Caveolin-1 is transported to multi-vesicular bodies after albumin-induced endocytosis of caveolae in HepG2 cells.

Caveolae-mediated endocytosis is a highly regulated endocytic pathway that exists in parallel to other forms of clathrin-dependent and -independent endocytosis. Internalized caveolae accumulate in intermediate organelles called caveosomes. Here we addressed the further fate of internalized caveolae by inducing caveolae-mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin-1 by immunogold labelling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Long-term (1 and 3 hrs) albumin treatment resulted in the appearance of albumin-containing caveolae in special multi-caveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome-like structures in the cytoplasm. In addition, numerous CD63 (LIMP-1) positive late endosomes/multi-vesicular bodies were found positive for caveolin-1, suggesting that upon albumin incubation, caveolin-1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long-term albumin uptake, caveolin-1 travels to late endosomes and is replaced by newly synthesized caveolin-1 at the plasma membrane.

Oesophageal tonsil of the chicken.

The oesophageal tonsil of the chicken is a novel member of the mucosal-associated lymphoid tissue (MALT), which is located around the entrance of the proventriculus. It consists of 6 to 8 single units, which are surrounded by a thin fibrous capsule. Each one is organised around the bottom of the longitudinal folds of the oesophagus, and serves as a 'tonsillar crypt'. Stratified squamous epithelium is infiltrated by lymphoid cells, i.e. T cells, plasma cells, macrophages, and dendritic cells, but not B cells, to form lymphoepithelium (LE). In the LE vimentin-, MHC II- and ATPase-positive cells possibly represent Langerhans' cells, but the appearance of 74.3 positive cells in the LE is unusual, because the 74.3 monoclonal antibody (mAb) recognises chicken follicular dendritic cells in the germinal centre and medulla of the bursal follicles. The subepithelial lymphoid tissue is organised into T- and B-dependent regions, which are the interfollicular areas and the germinal centres, respectively. Existence of high-endothelial venules in the interfollicular region suggests an extensive cellular connection between the oesophageal tonsil and the other lymphoid organs. In the resting oesophagus the lumen is closed, but during swallowing a bolus the crypt opens and the lymphoepithelium can be exposed to undigested food, antigens, infectious agents and vaccines. The location of the oesophageal tonsil, cranial to the stomach, may provide this organ with a unique role as compared to the other parts of the MALT; namely, it may contribute to the replication of infectious bursal disease virus (IBDV) and/or the pathogenesis of infectious bursal disease.

Origin of the bursal secretory dendritic cell.

The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick-quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected by double staining of vimentin plus 74.3 and vimentin plus QCPN monoclonal antibodies, respectively. In bursal primordia of 5- and 6-day-old quail embryos both dendritic cells and B cells were of host, i.e. chicken origin. Mixed dendritic cell population of quail and chick origin emerged in chimeric birds of 6.5 days of age. In quail embryos transplanted at 7 and 8 days of age both dendritic cells and B cells were mixed i.e. of chicken and quail origin. Bursal secretory dendritic cells and medullary epithelial cells create "dendro-epithelial tissue" to receive pre-B cells. Colonization of dendro-epithelial tissue by pre-B cells initiates at day 7, thus the colonization of bursal anlage by blood-borne cells is a two-step process; entering of dendritic cells at day 6.5 is followed by that of B cells at day 7 and afterwards. It is discussed that bursal secretory dendritic cells and their product are key elements of bursal function therefore the mammalian bursa equivalent organ might be represented by a cell, which is analogous with the bursal secretory dendritic cell.

Esophageal tonsil: a novel gut-associated lymphoid organ.

The esophageal tonsil of the chicken is a novel, significant element of the gut-associated lymphoid tissue (GALT). Its stable location and histological organization fulfills the meaning of the term "tonsil." The six-to-eight-isolated tonsillar units are located at the border of the esophagus and the proventriculus. The number of tonsillar units is identical with that of the esophageal folds. Each tonsillar unit consists of a crypt lined by lymphoepithelium and surrounded by dense lymphoid tissue, which is organized into T- and B-dependent regions, like peripheral lymphoid organs. The excretory ducts of the mucosal glands of the esophagus are frequently involved in the formation of the lymphoepithelium. The esophageal tonsil is anatomically located cranial to the stomach, unlike the other parts of the GALT. Therefore, it is continuously exposed to undigested environmental antigens, allergens, food, and infectious agents. To develop effective oral vaccines, the existence of the esophageal tonsil has to be taken into account.

Diverse expression of the K-1 antigen by cortico-medullary and reticular epithelial cells of the bursa of Fabricius in chicken and guinea fowl.

The immunocytochemical study of the K-1 monoclonal antibody indicates that the epithelial components of the bursa of Fabricius of the chicken and guinea fowl express the K-1 positive molecule. During embryogenesis, the K-1 antigen expression appears together with the bud-formation. As the number of B cells increases in the developing follicle, the K-1 expression gradually diminishes in the medullary reticular epithelial cells and completely ceases by hatching, which suggests that the molecule is developmentally regulated. After hatching, the expression of the molecule is restricted to the sealing off zone of the lymphoepithelial or medullary region of the follicle: i.e. to the cortico-medullary (CM) epithelial cells and the follicle associated epithelium (FAE) supporting cells in guinea fowl and to the latter ones in the chicken. The expression of the K-1 antigen by these epithelial components may support their structural identity. After hatching, the K-1 molecule is restricted to the CM epithelial cells and/or FAE supporting cells, which suggests that the function of the embryonic epithelial bud is taken over by the CM epithelial cells. The K-1 positive CM epithelial cells form arches, which encompass blast-like cells. The possible relationship of the CM epithelial cells and blast-like cells, which may represent the precursors of bursal secretory dendritic cells is discussed.

Ontogeny of the dendritic, and follicle-associated epithelial cells in the bursa of Fabricius of guinea fowl (Numida meleagris).

A panel of monoclonal antibodies was produced to study the mesenchymal stromal elements of the bursa of Fabricius in guinea hen. The intracellular antigens recognized by GIIF3 and NIC2 monoclonal antibodies are of 50 and 30 kD, respectively. The cells identified by these antibodies emerge in the mesenchyme around day 12 of incubation, and immigrate into the surface epithelium of the bursal folds, which precedes the follicle formation. The GIIF3 cells move up to the luminal surface of the follicle and differentiate to follicle-associated epithelial cells. The NIC2 cells remain in the medulla, produce and secrete large amount NIC2 positive substance, when the bursal function starts up. The presence of double positive (GIIF3 and NIC2) cells in the medulla around hatching, seems to indicate, that the two cells might have a common origin. The NIC2 positive product of the bursal secretory dendritic cells contributes to the microenvironment, and possibly necessary for the B cell clonal expansion and establisment of the immune repertoire in the guinea hen.

Differential expression of GL7 activation antigen on bone marrow B cell subpopulations and peripheral B cells.

GL7 was originally described as a 35-kDa late activation antigen on mouse T and B cells. GL7 expression has also been demonstrated on thymocytes, germinal center B cells and some neuronal cell types. Flow-cytometry and immunohistochemistry were used to follow changes in the expression of GL7 during B cell development, amongst B cell subpopulations and various anatomical locations. GL7 is expressed as early as the pro-B cell stage and increases up to the pre-B-I stadium. Expression remains high on pre-B-II and on immature B cells, although slightly decreases during maturation. GL7 is almost completely downregulated when IgD appears on the cell surface. On the periphery only a few B cells are positive and these cells are almost exclusively found in the sIgD- germinal center areas of lymph nodes and spleen. The staining pattern of GL7 is very similar to that of PNA in the lymph nodes but in the bone marrow we have found both B220+PNA+GL7- and B220+PNA+GL7+ populations, showing that GL7 and the antigen recognized by PNA are different. After in vitro stimulation, the GL7(hi) B cell population has also been found to be IgD negative. Functional comparison between in vitro activated and MACS sorted GL7(hi) and GL7(lo/-) spleen B cells of immunized mice showed significantly higher specific and total antibody production as well as antigen presenting capacity in the GL7(hi) population.

A novel monoclonal antibody identifies all avian embryonic myogenic cells and adult smooth muscle cells.

A novel monoclonal antibody, designated GIIF3, recognized prospective and differentiated smooth muscle cells in avian species studied - guinea fowl, chicken and quail. The GIIF3 antigen appeared in the myocardial, and the myotomal cells of the embryos at Hamburger-Hamilton stages 10 and 14, respectively. The expression of the GIIF3 molecule in the vascular smooth muscle cells emerged in the ventral wall of the dorsal aorta at Hamburger-Hamilton stage 16. The visceral smooth muscle cells started to produce the GIIF3 molecule from Hamburger-Hamilton stage 28 onwards. In both cardiac and skeletal muscles the GIIF3 expression gradually diminished, and it was lost by the end of the embryonic period, unlike in the differentiated vascular and visceral smooth muscle cells. In the latter cells the GIIF3 immunoreactive product showed a fine granular pattern that accumulated in the central region of the cytoplasm; it also occurred in the nucleus. A heavily stained discontinuous layer was associated with the cell membrane. The immunoblotting of the GIIF3 antibody recognized protein bands at 50 and 42 kDa in lysates of adult avian gizzard. A detailed comparative immunohistochemical study was made by smooth muscle markers, which confirmed the results of the immunoblotting, namely, that the GIIF3 monoclonal antibody recognized an avian myogenic cell specific molecule. During smooth muscle cell differentiation the GIIF3 molecule appeared as early as the alpha-smooth muscle actin, and in adult birds continued to be expressed; therefore the GIIF3 molecule could be regarded as a novel avian smooth muscle specific marker.

Functional restoration of the bursa of Fabricius following in ovo infectious bursal disease vaccination.

The primary role of the avian bursa of Fabricius is to provide an essential microenvironment for B-lymphocytes to diversify their immunoglobulin genes by gene hyperconversion. Infectious bursal disease (IBD) vaccination using intermediate plus vaccine strains can temporarily deplete the bursal follicles and interrupt the normal B-cell development, which is generally followed by B-cell repopulation and histological regeneration. To find evidence that functional restoration of the bursa of Fabricius occurs in addition to the histological regeneration, we have analysed the chB1 gene expression, which indicates active bursal B-lymphocytes, and also the surface expression of a carbohydrate structure Lewis(x), a marker which identifies those bursal B-lymphocytes that are undergoing gene hyperconversion. In ovo vaccination with an immune complex vaccine (IBDV-BDA) caused transient bursal destruction in both the SPF and the maternally protected broiler groups with differences evident in the starting time, the severity and the duration of the effect. After the depletion phase, signs of histological regeneration appeared together with chB1- and Lewis(x) expression indicating that B-lymphocytes were functionally active and the bursa of Fabricius was serving again as an efficient primary lymphoid organ providing an appropriate microenvironment for B-cell development.

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy.

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.