Lynn K. Gordon

Cleavage of pyrimidine dimers in specific DNA sequences by a pyrimidine dimer DNA-glycosylase of M. luteus

Pyrimidine dimer formation in response to UV radiation is governed by the thymine content of the potential dimer and the two flanking nucleotides. An enzymatic activity can be purified from Micrococcus luteus that cleaves the N-glycosyl bond between the 5′ pyrimidine of a dimer and the corresponding sugar without rupture of a phosphodiester bond. We propose that strand scission at a dimer site by the M. luteus enzyme requires two activities, a pyrimidine dimer DNA-glycosylase and an apyrimidinic/apurinic endonuclease.

Colonic Bacteria Express an Ulcerative Colitis pANCA-Related Protein Epitope

ABSTRACT Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae andEscherichia coli. Isolation and partial sequencing of theB. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.

Orbital inflammatory disease: a diagnostic and therapeutic challenge.

The spectrum of orbital inflammatory disease (OID) ranges broadly from specific disease diagnoses, for example, Wegeners granulomatosis or sarcoidosis, to nonspecific inflammation which may involve one or multiple structures of the orbit. Mimics of idiopathic OID must be considered in a comprehensive differential diagnosis and include malignancies, congenital mass lesions, infectious diseases, and occult or distant trauma. Idiopathic OID may be secondary to an underlying systemic inflammatory disease, which must be diagnosed in order to develop a comprehensive therapeutic plan, or may represent localized pathologic processes without systemic involvement. Evaluation of the patient with suspected OID must include a careful history, physical examination, directed laboratory, and radiologic studies, and may sometimes require tissue for diagnostic studies. Therapeutic options for inflammatory diseases are expanding as biologically targeted agents become available that act on specific segments of the inflammatory cascades. The purpose of this paper is to provide a framework for the evaluation and management of patients with the spectrum of diseases known as OID and to discuss some of the new advances in immunologic monitoring and targeted immune therapies that will likely play an increasingly important role in the care of these patients.

Ocular manifestations of multiple sclerosis.

Purpose of review Multiple sclerosis is an autoimmune demyelinating disorder of the nervous system that is commonly manifested by visual system involvement and that may initially present with ophthalmologic symptoms. This paper reviews recent findings regarding the ocular manifestations in multiple sclerosis. Recent findings Manifestations of multiple sclerosis in the eye include both the afferent and efferent visual pathways. Optic neuritis, the most common ocular manifestation of multiple sclerosis, may be the initial clinical disease manifestation. Recent long-term follow-up data show that most patients with demyelinating optic neuritis have an excellent prognosis for recovery of central visual acuity. Evidence is emerging, however, for significant and broad reduction in both contrast sensitivity and color perception in multiple sclerosis patients despite near-normal visual acuities. Ocular motor deficits in multiple sclerosis include internuclear ophthalmoplegia and nystagmus, resulting in diplopia, oscillopsia, blurred visual, loss of stereopsis, and reading fatigue. Multiple sclerosis also may be associated with ocular inflammatory diseases, in particular pars planitis and retinal periphlebitis. Summary Ocular findings may be initial manifestations of multiple sclerosis and may predict additional demyelinating events. Recognizing these syndromes and signs will help clinicians to properly evaluate the patient, formulate an appropriate differential diagnosis, be able to discuss the prognosis with the patient, and help develop an effective therapeutic plan.

Mesenchymal Stem Cell Population Derived from Human Pluripotent Stem Cells Displays Potent Immunomodulatory and Therapeutic Properties

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however, loss of potency and inconsistent quality severely limit their use. To overcome these issues, we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking, scraping, and sorting that other hESC-MSC derivation methods require. Moreover, unlike previous reports on hESC-MSCs, we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected, they dynamically secrete a range of bioactive factors, display enzymatic activity, and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However, they also display unique immunophenotypic properties, as well as a smaller size and >30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition, this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models, including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources.

Quantitation of cyclobutane pyrimidine dimer formation in double- and single-stranded DNA fragments of defined sequence.

The distribution of cyclobutane pyrimidine dimers in defined sequences of ultraviolet light-irradiated DNA was determined. The results demonstrate that the extent of dimer formation at a potential dimer site as a function of the dose reaches a steady-state level for all dimers at doses above 2000/Jm/sup 2/. The steady-state level is primarily dependent upon the composition of the dimer, varying from a maximum of about 10% dimer formation at sites of adjacent thymines to less than 1% for sites of adjacent cytosines. The extent of dimer formation is also affected by the two bases that immediately flank the potential dimer site as well as by longer-range sequence effects. The rates of dimer formation and the steady-state levels at most dimers are similar in single- and double-stranded DNA. The dose rate of irradiation does not affect the distribution of pyrimidine dimers over the range of 1.8-7.5 J/m/sup 2//sec. The implications of these observations for understanding mutation rates at different sites within a gene are discussed.

Molecular cloning of a Bacteroides caccae TonB-linked outer membrane protein identified by an inflammatory bowel disease marker antibody.

ABSTRACT Commensal enteric bacteria are a required pathogenic factor in inflammatory bowel disease (IBD), but the identity of the pertinent bacterial species is unresolved. Using an IBD-associated pANCA monoclonal antibody, a 100-kDa protein was recently characterized from an IBD clinical isolate of Bacteroides caccae (p2Lc3). In this study, consensus oligonucleotides were designed from 100-kDa peptides and used to identify a single-copy gene from the p2Lc3 genome. Sequence analysis of the genomic clone revealed a 2,844-bp (948 amino acid) open reading frame encoding features typical of the TonB-linked outer membrane protein family. This gene, termed ompW,was detected by Southern analysis only in B. caccae and was absent in other species of Bacteroides and gram-negative coliforms. The closest homologues of OmpW included the outer membrane proteins SusC of Bacteroides thetaiotaomicron and RagA of Porphyromonas gingivalis. Recombinant OmpW protein was immunoreactive with the monoclonal antibody, and serum anti-OmpW immunoglobulin A levels were elevated in a Crohns disease patient subset. These findings suggest that OmpW may be a target of the IBD-associated immune response and reveal its structural relationship to a bacterial virulence factor of P. gingivalis and periodontal disease.

Activation of autophagy in retinal ganglion cells

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell‐protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy‐related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule‐associated protein 1 light chain 3–II (LC3‐II) and beclin‐1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was up‐regulated 1.5‐ to 1.8‐fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6‐fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6‐, 2.7‐, and 1.7‐fold increases in LC3‐II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin‐1 was 1.7‐fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3‐methyladenine, and Wortmannin in RGC‐5 cells under serum‐deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC‐5 cells maintained under conditions of serum deprivation.

Doxycycline and intracranial hypertension

The authors report seven patients from six neuro-ophthalmology referral centers who developed pseudo-tumor cerebri during treatment with doxycycline. All four female patients and one of three male patients were obese. Vision was minimally affected in most patients, but two had substantial visual acuity or visual field loss at presentation. Discontinuation of doxycycline, with or without additional intracranial pressure-lowering agents, yielded improvement, but permanent visual acuity or visual field loss occurred in five patients.

Diagnostic dilemmas in orbital inflammatory disease

Orbital inflammatory disease (OID) broadly describes a variety of pathologic processes and clinical presentations. OID may be idiopathic or may be secondary to a systemic inflammatory disease, retained foreign body, or infectious disease. OID includes the spectrum of bacterial or fungal infections, diffuse inflammation of multiple tissues (e.g., sclerosing orbititis or diffuse anterior OID), and preferential involvement of specific orbital structures (e.g., orbital myositis or optic perineuritis). Mimics of OID include congenital orbital mass lesions or orbital neoplastic disease such as lymphoma or rhabdomyosarcoma. The ultimate diagnosis and treatment plan relies on a careful history and detailed clinical examination followed by the judicious use of ancillary diagnostic testing and a comprehensive treatment plan. The purpose of this review is to provide an overview of the spectrum of diseases known as OID, with emphasis on specific diagnostic challenges in the evaluation and management of patients with idiopathic OID.