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Background and Aim: Disease activity for Crohn's disease (CD) and UC is typically defined based on symptoms at a moment in time, and ignores the long-term burden of disease. The aims of this study were to select the attributes determining overall disease severity, to rank the importance of and to score these individual attributes for both CD and UC., Methods: Using a modified Delphi panel, 14 members of the International Organization for the Study of Inflammatory Bowel Diseases (IOIBD) selected the most important attributes related to IBD. Eighteen IOIBD members then completed a statistical exercise (conjoint analysis) to create a relative ranking of these attributes. Adjusted utilities were developed by creating proportions for each level within an attribute., Results: For CD, 15.8% of overall disease severity was attributed to the presence of mucosal lesions, 10.9% to history of a fistula, 9.7% to history of abscess and 7.4% to history of intestinal resection. For UC, 18.1% of overall disease severity was attributed to mucosal lesions, followed by 14.0% for impact on daily activities, 11.2% C reactive protein and 10.1% for prior experience with biologics. Overall disease severity indices were created on a 100-point scale by applying each attribute's average importance to the adjusted utilities., Conclusions: Based on specialist opinion, overall CD severity was associated more with intestinal damage, in contrast to overall UC disease severity, which was more dependent on symptoms and impact on daily life. Once validated, disease severity indices may provide a useful tool for consistent assessment of overall disease severity in patients with IBD., Competing Interests: Competing interests: Phase 1 was funded thanks to an unrestricted educational grant to IOIBD from AbbVie and Tillotts. CAS has served as a consultant on advisory boards for AbbVie, Amgen, Lilly, Janssen, Sandoz, Pfizer, Prometheus, Takeda, Theradiag and UCB; as a speaker for American Regent, AbbVie, Janssen, Pfizer and Takeda; and receives grant support from AbbVie, Janssen and Takeda. BMRS has research grants from Shire and Takeda. BF has served as a consultant for Abbott/AbbVie, Actogenix, Akros, Albireo Pharma, Amgen, Astra Zeneca, Avaxia Biologics, Avir Pharma, Axcan, Baxter Healthcare, Biogen Idec, Boehringer-Ingelheim, Bristol-Myers Squibb, Calypso Biotech, Celgene, Elan/Biogen, EnGene, Ferring Pharma, Roche/Genentech, GiCare Pharma, Gilead, Given Imaging, GSK, Ironwood Pharma, Janssen Biotech (Centocor), JnJ/Janssen, Kyowa Kakko Kirin Co., Lexicon, Lilly, Lycera BioTech, Merck, Mesoblast Pharma, Millennium, Nektar, Nestles, Novonordisk, Pfizer, Prometheus Therapeutics and Diagnostics, Protagonist, Receptos, Salix Pharma, Serono, Shire, Sigmoid Pharma, Synergy Pharma, Takeda, Teva Pharma, TiGenix, Tillotts, UCB Pharma, Vertex Pharma, VHsquared, Warner-Chilcott, Wyeth, Zealand and Zyngenia; has been a speaker for Abbott/AbbVie, JnJ/Janssen, Takeda, Warner-Chilcott and UCB Pharma; has served on advisory boards for Abbott/AbbVie, Amgen, Astra Zeneca, Avaxia Biologics, Bristol-Myers Squibb, Celgene, Centocor, Elan/Biogen, Ferring, JnJ/Janssen, Merck, Nestles, Novartis, Novonordisk, Pfizer, Prometheus Laboratories, Protagonist, Salix Pharma, Takeda, Teva, TiGenix, Tillotts Pharma AG and UCB Pharma; receives research funding from Abbott/AbbVie, Amgen, Astra Zeneca, Bristol-Myers Squibb (BMS), Janssen Biotech (Centocor), JnJ/Janssen, Roche/Genentech, Millennium, Pfizer, Receptos, Santarus, Sanofi, Tillotts and UCB Pharma; and serves on the board of directors for Robarts Clinical Trials. BS has served as a consultant for AbbVie, Celgene, Janssen Biotech, MedImmune, Takeda, Pfizer, TiGenix, Bristol-Myers Squibb, Receptos, Akros Pharma, Arena Pharmaceuticals, Theravance Biopharma R&D, Boehringer-Ingelheim, Synergy Pharmaceuticals, Toplvert Pharma, UCB and Lilly; has served on scientific advisory boards for Celgene, Janssen Biotech, MedImmune, Takeda, Pfizer, TiGenix and Lily; and he receives research funding from Celgene, Janssen Biotech, MedImmune, Takeda and Pfizer. EVL has consulted for UCB Pharma, Takeda, Janssen, AbbVie, Genentech, Celgene, Amgen, Mesoblast, Theradiag, Sun Pharma and Seres Health; has received research support from UCB Pharma, Takeda, Janssen, AbbVie, Genentech, Celgene, Amgen, Pfizer, Receptos, Gilead and Robarts Clinical Trials. RP has served as a consultant for Abbvie/Abbott, Amgen, Aptalis, AstraZeneca, Baxter, Eisai, Ferring, Janssen, Merck, Schering-Plough, Shire, Centocor, Elan, Glaxo-Smith Kline, UCB, Pfizer, Bristol-Myers Squibb, Warmer Chilcot, Takeda, Cubist and Celgene; on speaker's bureaus for Abbvie/Abbott, AstraZeneca, Janssen, Schering-Plough, Shire, Centocor, Elan, Prometheus, Warner Chilcott and Takeda; on advisory boards for Abbvie/Abbott, Amgen, Aptalis, AstraZeneca, Baxter, Eisai, Ferring, Genentech, Janssen, Merck, Schering-Plough, Shire, Centocor, Elan, Glaxo-Smith Kline, UCB, Pfizer, Bristol-Myers Squibb, Warner Chilcott, Takeda, Cubist, Celgene and Salix; and receives research/educational support from Abbvie, Abbott, Ferring, Janssen, Schering-Plough, Centocor, Millenium, Elan, Proctor and Gamble and Bristol-Myers Squibb. GD has served as advisor for Abbvie, Ablynx, Amakem, AM Pharma, Avaxia, Biogen, Bristol-Meyers Squibb, Boerhinger Ingelheim, Celgene, Celltrion, Cosmo, Covidien, Ferring, DrFALK Pharma, Engene, Galapagos, Gilead, Glaxo Smith Kline, Hospira, Johnson & Johnson, Medimetrics, Millenium/Takeda, Mitsubishi Pharma, Merck Sharp Dome, Mundipharma, Novonordisk, Pfizer, Prometheus laboratories/Nestle, Receptos, Robarts Clinical Trials, Salix, Sandoz, Setpoint, Shire, Teva, Tigenix, Tillotts, Topivert, Versant and Vifor; and received speaker fees from Abbvie, Ferring, Johnson & Johnson, Merck Sharp Dome, Mundipharma, Norgine, Pfizer, Shire, Millenium/Takeda, Tillotts and Vifor. CNB is supported in part by the Bingham Chair in Gastroenterology. He has served on advisory boards to Abbvie Canada, Janssen Canada, Shire Canada, Takeda Canada and Pfizer Canada and has consulted to Mylan Pharmaceuticals; he has received unrestricted educational grants from Abbvie Canada, Janssen Canada, Shire Canada and Takeda Canada. RG has received consulting and speaking fees from AbbVie, Janssen, MSD, Ferring, Takeda and Baxter; and research grants from AbbVie. GJM has served as speaker or advisory board member for AbbVie, Angelini, Astellas, Danone, MSD, Falk Pharma, Ferring, Hospira, Janssen, Omega Pharma, Otsuka, Pharmacosmos, Pfizer, Sandoz and Takeda; as consultant for Janssen, MSD, Takeda and Omega Pharma; and has received research grants in the last 3 years from Menarini, AbbVie, and MSD. BS has served on advisory boards for Dann and Yakult North American Probiotic Council, Second Genome, Lilly and Enterome; and receives grant support from Janssen, Salix and GSK. MSS has received consulting and speaker fees from AbbVie, Amgen, Ferring, Janssen, Merck, Pfizer, Prometheus, Shire and Takeda; and research funding from AbbVie, Janssen, Prometheus and Takeda. IEK has served as a consultant and on advisory boards for AbbVie and MSD. CO is the principal investigator for Redhill Pharma. DPBM is a consultant for UCB, Jannsen, Merck and Second Genome. WR has served as a speaker for Abbott Laboratories, Abbvie, Aesca, Aptalis, Centocor, Celltrion, Danone Austria, Elan, Falk Pharma GmbH, Ferring, Immundiagnostik, Mitsubishi Tanabe Pharma Corporation, MSD, Otsuka, PDL, Pharmacosmos, Schering-Plough, Shire, Takeda, Therakos, Vifor and Yakult; as a consultant for Abbott Laboratories, Abbvie, Aesca, Amgen, AM Pharma, Astellas, Astra Zeneca, Avaxia, Bioclinica, Biogen IDEC, Boehringer-Ingelheim, Bristol-Myers Squibb, Cellerix, Chemocentryx, Celgene, Centocor, Celltrion, Covance, Danone Austria, Elan, Falk Pharma GmbH, Ferring, Galapagos, Genentech, Gilead, Grünenthal, ICON, Index Pharma, Inova, Janssen, Johnson & Johnson, Kyowa Hakko Kirin Pharma, Lipid Therapeutics, MedImmune, Millenium, Mitsubishi Tanabe Pharma Corporation, MSD, Nestle, Novartis, Ocera, Otsuka, PDL, Pharmacosmos, Pfizer, Procter & Gamble, Prometheus, Robarts Clinical Trial, Schering-Plough, Second Genome, Setpointmedical, Takeda, Therakos, Tigenix, UCB, Vifor, Zyngenia and 4SC; as an advisory board member for Abbott Laboratories, Abbvie, Aesca, Amgen, AM Pharma, Astellas, Astra Zeneca, Avaxia, Biogen IDEC, Boehringer-Ingelheim, Bristol-Myers Squibb, Cellerix, Chemocentryx, Celgene, Centocor, Celltrion, Danone Austria, Elan, Ferring, Galapagos, Genentech, Grünenthal, Inova, Janssen, Johnson & Johnson, Kyowa Hakko Kirin Pharma, Lipid Therapeutics, MedImmune, Millenium, Mitsubishi Tanabe Pharma Corporation, MSD, Nestle, Novartis, Ocera, Otsuka, PDL, Pharmacosmos, Pfizer, Procter & Gamble, Prometheus, Schering-Plough, Second Genome, Setpointmedical, Takeda, Therakos, Tigenix, UCB, Zyngenia and 4SC; and has received research funding from Abbott Laboratories, Abbvie, Aesca, Centocor, Falk Pharma GmbH, Immundiagnsotik and MSD. GR has consulted to Abbot, Abbvie, Augurix, Boehringer, Calypso, FALK, Ferring, Fisher, Genentech, Essex/MSD, Novartis, Pfizer, Phadia, Roche, UCB, Takeda, Tillots, Vifor, Vital Solutions and Zeller; has received speaker's honoraria from Astra Zeneca, Abbott, Abbvie, FALK, MSD, Phadia, Tillots, UCB and Vifor; has received educational grants and research grants from Abbott/Abbvie, Ardeypharm, Augurix, Calypso, Essex/MSD, FALK, Flamentera, Novartis, Roche, Takeda, Tillots, UCB and Zeller. WK has received fees for advising from Dr. Falk Pharma GmbH, Ferring International, GA-Analysis; support for research from Dr. Falk Pharma GmbH and GA-Analysis; grants for lectures from Abbvie, Ardeypharm, Falk Foundation, Ferring Arzneimittel, GA-Analysis, Institut Allergosan, Nikkiso, Otsuka and Recordati. CT has served as a speaker for Dr. Falk Pharma, Tillotts Pharma, Ferring, MSD and AstraZeneca. SS has received consulting fees from AbbVie, Boehringer; Celltrion/Mundipharma, Jansen, Novartis, Merck, Pfizer/Hospira, Sanofi, Takeda and UCB; and speaking fees from AbbVie Ferring, Falk, Merck, Takeda and Shire. SD has served as a speaker, a consultant and an advisory board member for Abbvie, Ferring, Hospira, Johnson & Johnson, Merck, Millennium Takeda, Mundipharma, Pfizer, Tigenix, UCB Pharma and Vifor. WS reports grant support from Receptos, Exact Sciences, Amgen, the American College of Gastroenterology and the Broad Foundation; grant support and personal fees from Prometheus Laboratories, AbbVie, Boehringer Ingelheim, Takeda, Atlantic Pharmaceuticals, Janssen, Bristol-Myers Squibb, Genentech, Pfizer and Nutrition Science Partners; and personal fees from Kyowa Hakko Kirin, Millennium Pharmaceuticals, Celgene Cellular Therapeutics, Santarus, Salix Pharmaceuticals, Catabasis Pharmaceuticals, Vertex Pharmaceuticals, Warner Chilcott, Gilead Sciences, Cosmo Pharmaceuticals, Ferring Pharmaceuticals, Sigmoid Biotechnologies, Tillotts Pharma, Am Pharma BV, Dr. August Wolff, Avaxia Biologics, Zyngenia, Ironwood Pharmaceuticals, Index Pharmaceuticals, Nestle, Lexicon Pharmaceuticals, UCB Pharma, Orexigen, Luitpold Pharmaceuticals, Baxter Healthcare, Ferring Research Institute, Amgen, Novo Nordisk, Mesoblast, Shire, Ardelyx, Actavis, Seattle Genetics, MedImmune (AstraZeneca), Actogenix NV, Lipid Therapeutics Gmbh, Eisai, Qu Biologics, Toray Industries, Teva Pharmaceuticals, Eli Lilly, Chiasma, TiGenix, Adherion Therapeutics, Immune Pharmaceuticals, Celgene, Arena Pharmaceuticals, Ambrx, Akros Pharma, Vascular Biogenics, Theradiag, Forward Pharma, Regeneron, Galapagos, Seres Health, Ritter Pharmaceuticals, Theravance, Palatin, Biogen and the University of Western Ontario (owner of Robarts Clinical Trials). AG has served as a consultant for AbbVie, Janssen, Merck and Takeda; a speaker for Janssen; and received research and clinical programme support from AbbVie and Janssen. ST has received Grants/Research Support from AbbVie, IOIBD, Lilly, UCB, Vifor and Norman Collison Foundation; consulting fees from AbbVie, Amgen, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Chemocentryx, Cosmo, Ferring, Giuliani SpA, GlaxoSmithKline, Lilly, MSD, Neovacs, NovoNordisk, Norman Collison Foundation, Novartis, NPS Pharmaceuticals, Pfizer, Proximagen, Receptos, Shire, Sigmoid Pharma, Takeda, Topivert, UCB, VHsquared and Vifor; speaker fees from AbbVie, Ferring and Takeda. JP has received consultant fees from Abbvie, Boehringer Ingelheim, Celltrion, Galapagos, Genentech-Roche, Janssen, Pfizer, Takeda, TiGenix and Topivert; speaker fees from Abbvie, Celltrion, Janssen, MSD and Pfizer. JFC has served as a consultant or advisory board member for AbbVie, Amgen, Boehringer-Ingelheim, Celgene Corporation, Celltrion, Enterome, Ferring, Genentech, Janssen and Janssen, Medimmune, Merck & Co., Pfizer, Protagonist, Second Genome, Seres, Takeda and Theradiag; a speaker for AbbVie, Ferring, Takeda and Shire; receives research support from Abbvie, Janssen and Janssen, Genentech and Takeda; and has stock options for Intestinal Biotech Development and Genfit. SH is a consultant for AbbVie, Actavis, Amgen, Arena, Astellas Pharma Global, Astra Zeneca, Baxter, Boehringer Ingelheim, Bristol Myers Squibb, Catabasis, Cellgene, Celltrion, Cubist, Ferring, Forest Labs, Genentech, Glenmark, GSK, Hospira, Janssen, Lilly, Lutipold/American Regent, Meda, Nestle, Novartis, Novo Nordisk, Pfizer, Prometheus, Receptos, Salix, Sanofi-Avantis, Seattle-Genetics, Seres Health, Shire, Takeda, Theradiag, Tigenex, UCB Pharma and VHsquared; does clinical research with Abbvie, Amgen, Genentech, GSK, Janssen, Lilly, Lutipold/American Regent, Novartis, Novo Nordisk, Pfizer, Prometheus, Receptos, Sanofi-Avantis, Takeda and UCB Pharma; is a speaker for AbbVie, Janssen and Takeda; and serves on a DSMB for Bristol Myers Squibb. LPB reports consulting fees from Merck, Abbvie, Janssen, Genentech, Mitsubishi, Ferring, Norgine, Tillots, Vifor, Therakos, Pharmacosmos, Pilège, BMS, UCB-pharma, Hospira, Celltrion, Takeda, Biogaran, Boerhinger-Ingelheim, Lilly, Pfizer, HAC-Pharma, Index Pharmaceuticals, Amgen, Sandoz, Forward Pharma GmbH, Celgene, Biogen, Lycera and Samsung Bioepis; and lecture fees from Merck, Abbvie, Takeda, Janssen, Takeda, Ferring, Norgine, Tillots, Vifor, Therakos, Mitsubishi and HAC-pharma., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

As with many clinical studies, trials using probiotics have shown clearly that some patients benefit from the treatment while others do not. For example if treatment with probiotics leads to 36% cure rate of diarrhea, why did the other 64% not have the same result? The issue is important for human and indeed experimental animal studies for two main reasons: (i) Would changing the design of the study result in more subjects responding to treatment? (ii) If a subject does not respond what are the mechanistic reasons? In order to tackle the issue of responders and non-responders to therapy, a workshop was held by the International Scientific Association for Probiotics and Prebiotics (ISAPP). The outcome was four recommendations. 1. Clearly define the end goal: this could be supporting a health claim or having the highest clinical effect and impact. 2. Design the study to maximize the chance of a positive response by identifying precise parameters and defining the level of response that will be tested. 3. Base the selection of the intervention on scientific investigations: which strain(s) and/or product formulation should be used and why. 4. Carefully select the study cohort: use biological or genetic markers when available to stratify the patient population before enrollment and decide at what point intervention will provide the best outcome (for example, in acute phase of disease, or during remission, with or without use of pharmaceutical agents). By following these recommendations and selecting an appropriate primary outcome, it is hoped that clinical data will emerge in the future that expands our knowledge of which probiotics benefits which subjects and by what mechanism.

Recent studies suggest that p16 overexpression determined by immunohistochemistry would be a specific marker for neoplastic and dysplastic squamous cells associated with high-risk HPV. The purpose of this study was to assess the correlation between cyto-histological findings, p16 expression and HPV subtype. A total of 99 biopsies were selected, 4 endocervical curettages and 95 uterine cervix biopsies, as well as 30 cervicovaginal smears from other 30 patients. The samples were divided according to the morphological diagnosis. Paraffin-embedded sections and cervicovaginal smears were immunostained using the CINtec p16INK4a Cytology Kit (DAKO). HPV was analyzed by PCR in 25 of the 99 biopsies with low-grade squamous intraepithelial lesion. Among those patients with neither HPV nor dysplasia, 1 of 35 (2.9%) biopsies and 1 of 11 (9%) smears were positive for p16. Sixteen of 25 (64%) biopsies and 6 of 10 (60%) smears of the low-grade lesion cases, and 38 of 39 (97.4%) biopsies and 8 of 9 (89%) smears of the high-grade lesion and squamous carcinoma were positive for p16. All cases of HPV-6/11 were negative or focally positive for p16. Most cases of HPV-18 or other subtypes were diffusely positive. Our results indicate that p16 expression is highly correlated with cyto-histological diagnosis, and is associated with diffuse staining and high-risk HPV. This technique provides greater objectivity in doubtful cases, and helps select patients at risk of disease progression at an acceptable cost when used in large populations.

Specific pathogen free (SPF) rodents are derived from germfree animals that are colonized with Schaedler's flora, a cocktail of eight bacterial strains isolated from the natural biota of mice. During successive generations SPF animals acquire a complex biota, but it is not known how similar it is to natural mouse biota. Therefore, fecal pellets of two feral mice and three SPF mice were studied by small subunit ribosomal DNA sequence analysis. After amplification of 16S rDNA by Bacterial Kingdom-specific primers, 132 rDNA clones from feral mice and 219 clones from SPF mice were placed phylogenetically. Forty-four percent of recovered rDNAs from feral mice were from organisms belonging to the Ribosomal Database Project's Bacteroides Group with significant proportions also coming from lactobacilli, the Clostridium coccoides Group and the Clostridium leptum Group. Although the SPF biota appeared equally complex at lower phylogenetic levels, the major phylogenetic groups represented were less diverse in that 92% of rDNA's from SPF mice mapped to groups of clostridia with 79% to the C. coccoides Group alone. Given the number of physiological parameters influenced by the gut biota and the importance of mice in biomedical research, further investigations are warranted.

Objective: To evaluate the changes induced in silver-stained nucleolar organizer regions (AgNORs) by the first fraction of a radiotherapy protocol for gynecologic cancer on exfoliated cytologic samples to predict the therapeutic success of the full protocol., Study Design: Thirteen gynecologic cancer patients who were scheduled for radiotherapy were included in the study. Cell smears were taken from the affected area before and after the first fraction of a radiotherapy protocol and silver stained for AgNORs. AgNORs per nucleus were counted under a light microscope. Local disease control by the full radiotherapy protocol was assessed at one year by the Papanicolaou technique., Results: Local success of radiotherapy was greater for lesions with higher pretreatment AgNOR counts and for lesions that underwent a greater percentage reduction in AgNOR counts after the first fraction. We correlated local success of the full radiotherapy protocol with a predictive index based on AgNOR counts obtained before and after the first fraction., Conclusion: A predictive index based on AgNOR counts can predict, as early as after the first fraction, the local control of disease by a full radiotherapy protocol. Knowledge of the probability of success long before the protocol is completed would allow reevaluation of therapeutic options.

Objectives: Chronic otitis media with effusion (COME) is the most prevalent inflammatory disease in children and is associated with numerous adverse long-term sequelae. Many factors have been associated with an increased risk of developing COME, one of which may be a genetic predisposition to the disease. To study the role that genetics play in the pathogenesis of COME, we used an animal model to compare the middle ear inflammatory responses in two different strains of rats (Lewis and Fisher)., Methods: In earlier studies, we demonstrated that exposure of the middle ear to endotoxin caused early extensive exudation and, later, goblet cell hyperplasia and mucin hypersecretion. In the present study, the animals were divided into six groups. In each group the animals were given transtympanic injection with gram-positive bacterial cell wall product (peptidoglycan-polysaccharide [PG-PS]). The middle ear bullae were studied at 1 week and 3 weeks after infection, and after systemic reinfection. Comparisons were made of the quantity of mucin exudate by enzyme-linked immunosorbent assay and by histological evaluation of the middle ear epithelial thickness., Results: Our data demonstrate a statistically significant difference in middle ear inflammation and effusion formation between the two genetically different strains of rats., Conclusions: These data support the hypothesis that the middle ear response to PG-PS may be genetically determined and therefore suggest that genetic predisposition may play a role in the pathogenesis of COME.

Objective: This study addresses the interaction of bacterial antigens, specifically peptidoglycan-polysaccharide (PG-PS) and lipopolysaccharide (LPS), in the induction and reactivation of mucoid middle ear effusions., Methods: Twenty-seven rats underwent eustachian tube obstruction before inoculation of the middle ear bulla with PG-PS. Three weeks later, after resolution of all middle ear effusions, 6 rats were randomly selected and euthanized as the first control group (control I). The remaining 21 animals were randomly assigned to 3 groups that received intravenous injections of Krebs Ringer (control II), PG-PS, and LPS, respectively. These rats were euthanized 2 days after intravenous injection. Middle ear mucin production and histologic changes were measured in all animals., Results: The mean concentrations of mucin were 0.94 +/- 0.52 mg/mL, 0.41 +/- 0.87 mg/mL, 16.33 +/- 3.67 mg/mL, and 1.15 +/- 0.41 mg/mL in the control I, control II, PG-PS, and LPS groups, respectively. Thus the mean concentration of mucin in the middle ear lavage samples was significantly greater in rats that were injected intravenously with PG-PS than in rats in other groups (P < 0.05). Histologic analyses demonstrated a greater degree of goblet cell hyperplasia in the PG-PS group than in other groups., Conclusions: This is the first animal model of recurring otitis media with effusion in which a systemic injection of PG-PS was used to reactivate a middle ear effusion in rats previously primed with a transtympanic injection of PG-PS. This study suggests that after otitis media with effusion has resolved, it may be reactivated by the presence of bacterial antigens and/or cytokines in the systemic circulation.

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.

: We report our initial characterization of the immune system in C3H/HeJBir mice that spontaneously develop perianal ulceration. Analysis of lymphokine production by mucosal and systemic lymphoid cells obtained from these mice revealed higher levels of interleukin (IL)4 in supernatants of in vitro stimulated Peyer's patch and mesenteric lymph node cells from C3H/HeJBir mice with severe perianal lesions than from C3H/HeJBir mice with mild perianal lesions or from normal C3H/HeJ mice. Supernatants that contain the highest amounts of IL-4 contain the lowest amounts of interferon (IFN)-γ. Peyer's patches and mesenteric lymph nodes of C3H/HeJBir mice with severe perianal lesions contain approximately four-fold fewer cells; however, inguinal lymph nodes contain up to 25-fold more cells compared with C3H/HeJ mice. These inguinal lymph node cells secrete IL-6, and T cells that produce IL-2 or IFN-γ can be demonstrated. Serum IgE, IgGl, and IgG2a are all increased in C3H/HeJBir mice with severe perianal ulceration. These results show that C3H/HeJBir mice with grossly evident inflammation have activated lymphocytes in both mucosal and systemic lymphoid organs. Understanding mechanisms of lymphocyte activation and regulation in these mice may provide insights into the pathogenesis of perianal ulceration that is often associated with Crohn's disease.

We have studied the localizations of transforming growth factor-beta (TGF-beta) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-beta 3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-beta 3 mRNA as well as TGF-beta 2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-beta 2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-beta 2 and 3 until E11, but even then the moderate signals for TGF-beta 3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-beta 3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-beta receptor type II, and treatment of these cells with NGF induces expression of TGF-beta 3 mRNA. The TGF-beta isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-beta isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-beta 2 and -beta 3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-beta s in fundamental events of neural development.

Estrogenic stimulation is a potent risk factor for the development of uterine cancer. More recently, analysis of patients in prospective breast cancer trials have established that tamoxifen also increases uterine cancer risk. In this report, uteri of oophorectomized rats were examined to ascertain the effects of estrogen and tamoxifen on the uterine induction of two isoforms of transforming growth factor-beta (TGF-beta). In contrast to studies of cells derived from breast epithelium, our studies reveal that both estrogen and tamoxifen increase immunoreactive TGF-beta. These changes were particularly pronounced in the endometrial stroma. Effects of progesterone also were examined and found to be distinct and relatively restricted to the glandular epithelium. These studies indicate that, in the uteri of oophorectomized rats, tamoxifen exerts estrogen-like effects on a peptide previously implicated in the control of cellular growth and differentiation. We hypothesize that induction of TGF-beta isoforms may be an important mediator of both estrogen- and tamoxifen-induced proliferative disorders in the uterus.

Pyoderma gangrenosum (PG) is a debilitating skin disease most often associated with inflammatory bowel disease and is a reportedly rare cause of peristomal ulceration. The lesions of PG rapidly evolve from small, erythematous pustules to deep, painful, pyogenic ulcers within hours to days of onset. Although the behavior and the appearance of the lesions of peristomal PG are diagnostic, a lack of familiarity with PG often leads to misdiagnosis and inappropriate therapy. This study reports four cases of peristomal PG and discusses the 20 previously reported cases in patients with inflammatory bowel disease. Seventy-five percent of patients were female and 67% had Crohn's disease. All patients had colitis, including all of the patients with Crohn's disease, 82% of whom had additional perineal complications. The diagnosis of peristomal PG was based on clinical appearance alone in 83% of cases. The onset of peristomal PG ranged from 2 weeks to 3 years following ostomy. The response to medical therapy was variable. All cases (17 of 17) treated with high-dose corticosteroids and local wound care responded, but five cases required additional therapy. No patient was successfully treated with stoma revision. Risk factors for the development of peristomal PG include Crohn's colitis, female gender, and perineal disease. While most patients respond well to systemic steroids and local wound care, up to one third of patients require long-term medical management.