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Biomedical subjects

L K Gordon

Publications and source records attributed to L K Gordon.

At least 19 recordsLinked to original sources

Orbital inflammatory disease: a diagnostic and therapeutic challenge.

The spectrum of orbital inflammatory disease (OID) ranges broadly from specific disease diagnoses, for example, Wegener's 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.

Diagnosis, Differential↗

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.

Acne Vulgaris↗

Role of extracellular peroxidase in the superoxide production by wheat root cells.

Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN3 and activated by MnCl2 and H2O2. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H2O2-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extra-cellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.

Adrenochrome↗

Ocular pANCA antigens are expressed in nonpigmented ciliary body epithelium and are conserved in multiple mammalian species.

PURPOSE: pANCA marker autoantibody is expressed by a subset of patients with anterior uveitis. A recombinantly isolated pANCA monoclonal antibody, Fab 5-3, identifies ocular expression of corresponding pANCA antigens in human ciliary body and retina. In this study, Fab 5-3 was used to explore pANCA antigen expression in ocular tissues of multiple mammalian species and identify the ciliary body cell type expressing the pANCA antigen. METHODS: Ocular tissues were obtained from several mammalian species and evaluated for expression of the pANCA (Fab 5-3) antigen(s) using immunohistochemistry and Western analysis of tissue extracts. Additionally, primary cultures of nonpigmented and pigmented rabbit ciliary body epithelium were analyzed for pANCA expression using immunofluorescence and Western analysis. RESULTS: Ocular pANCA (Fab 5-3) antigen expression was observed by immunohistochemistry only in the cytoplasm of retinal ganglion cells and ciliary body epithelium. Retinal antigen expression was conserved in all species examined. Ciliary body expression was observed in human, rabbit, rat, and mouse, but not in pig or cow. Antigen expression in the rabbit ciliary body was restricted to the nonpigmented layer as defined in primary cultures of nonpigmented and pigmented ciliary body epithelium. Immunoreactive proteins in both the human and rabbit included a 32-33 kDa doublet (histone H1), and novel 80 and 100 kDa proteins. CONCLUSIONS: This study identifies ocular pANCA antigen expression in multiple mammalian species localized to the retinal ganglion cell layer and the non-pigmented ciliary body epithelium. The present study also establishes novel 80 and 100 kDa proteins which may correspond to the cytoplasmic antigens detected in situ and can be further characterized biochemically and immunologically using small animal model systems.

Animals↗

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

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 Crohn's 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.

Amino Acid Sequence↗

Mast cell and neuroendocrine cytoplasmic autoantigen(s) detected by monoclonal pANCA antibodies.

pANCA is a marker antibody expressed in most patients with ulcerative colitis, and its cognate antigen is potentially an immunologic target in this disease. This study evaluates whether pANCA detects an autoantigen that is expressed in the colonic mucosa. Immunohistochemistry of colon specimens with human pANCA monoclonal antibodies (Fab 5-2 and 5-3) revealed a minor population of immunoreactive mucosal cells bearing a cytoplasmic vesicle antigen. By distribution, morphology, and tryptase expression, these were identified as mast cells. Immunofluorescent analysis revealed similar immunoreactivity of mouse mast cell lines and human KU812. Western analysis of mouse mast cell lines revealed immunoreactive proteins, and these were distinct from previously proposed pANCA antigens (histone H1, HMG 1 and 2, and neutrophil vesicle antigens). Cognate antigen for Fab 5-2 and 5-3 was also expressed in other tissue mast cells, cerebellar neurons, and pancreatic islet cells. These findings identify a novel cytoplasmic autoantigen(s) associated with UC by its presence in colonic mucosa and recognition by a disease-associated marker antibody.

Animals↗

Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA.

Perinuclear anti-neutrophil cytoplasmic antibody (pANCA)(4)is a predominant serum marker of ulcerative colitis (UC), and a familial trait associated with disease susceptibility and disease associated MHC haplotypes. This study characterizes the pANCA antigen defined by representative UC-pANCA human monoclonal antibodies, Fab 5-3 and 5-2. Western blot analysis probed with Fab 5-3 revealed specific binding to a nuclear protein doublet (apparent MW=32-33 kDa) expressed in several cell types. Purification and tryptic peptide sequencing identified the protein as histone H1, and this specificity was confirmed by Fab 5-3 binding to purified H1. Rabbit anti-histone H1 immunostaining and Western blot analysis confirmed that the pANCA epitope is preferentially immunoaccessible in polymorphonuclear neutrophils (PMN). The epitope was localized to the COOH-terminal region by site-specific proteolysis, and recombinant deletants further localized binding activity for both Fab 5-2 and 5-3 to two non-overlapping segments (AA 69-171 and 172-226) associated with a recurring PKKAK motif. Serum IgG binding was detectable to these segments, but was not significantly correlated with pANCA titer or disease status. These findings indicate that histone H1 bears a recurring COOH-terminal epitope recognized by monoclonal ulcerative colitis-associated pANCA marker antibodies, but this epitope is not a predominant specificity of serum pANCA.

Amino Acid Sequence↗

Colonic bacteria express an ulcerative colitis pANCA-related protein epitope.

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 and Escherichia coli. Isolation and partial sequencing of the B. 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.

Amino Acid Sequence↗

Definition of ocular antigens in ciliary body and retinal ganglion cells by the marker antibody pANCA.

PURPOSE: A subset of patients with anterior uveitis express the marker, perinuclear anti-neutrophil cytoplasmic antibody (pANCA). In this study, recombinantly isolated pANCA monoclonal antibodies were used to search for ocular cells expressing the pANCA antigen. METHODS: Paraffin sections of human ocular tissues obtained after death were analyzed by immunohistochemistry to identify cell types expressing pANCA antigen. Microdissected eye-bank ocular tissue was characterized by western blot analysis to confirm antigen expression and identify candidate protein species. RESULTS: Immunohistochemical analysis with pANCA monoclonal antibodies revealed cytoplasmic antigen expression in retinal ganglion cells and ciliary body epithelium. pANCA antigen expression was restricted to tissues bearing these cell types by western blot analysis. A common set of epitope-positive protein species was shared by the two tissues (28 kDa, 80 kDa, and 90 kDa). Comparison of ocular tissues from seven subjects revealed no heterogeneity in antigen expression. CONCLUSIONS: In this study, novel cytoplasmic antigens of the pANCA marker antibody expressed in ciliary body and retinal tissue were identified. Validation of these antigens as targets of inflammation in pANCA+ uveitis requires further biochemical and immunologic analysis.

Antibodies, Antineutrophil Cytoplasmic↗

pANCA antibodies in patients with anterior uveitis: identification of a marker antibody usually associated with ulcerative colitis.

A pANCA autoantibody (antineutrophil cytoplasmic antibody, perinuclear pattern) has been described in uveitis patients, but its correlation with systemic illnesses and the specific type of pANCA has not been defined. The goals of this study were to determine the (1) frequency of pANCA autoantibodies in uveitis, (2) systemic associations in the pANCA + uveitis patients, and (3) type of pANCA antigen recognized by the uveitis-associated autoantibody. Serum was obtained from 59 patients with anterior uveitis or panuveitis and from nonuveitis controls. A detailed medical and family history was obtained from each subject at the time of phlebotomy. Sera were screened by neutrophil ELISA to determine the frequency of ANCA positivity. Immunofluorescence assays were then used to differentiate cANCA from pANCA. The specificity of the pANCA + antibodies was further characterized by DNase 1 sensitivity and granule antigen ELISAs. ANCA antibodies were detected in 29% of all patients with panuveitis or anterior uveitis. In 41% of these ANCA + patients, serum antibody detected a perinuclear antigen that was sensitive in all cases to DNase 1. The majority of pANCA + uveitis patients were either HLA-B27 positive or had systemic evidence of immune-mediated diseases. Two pANCA + patients had no medical or family history of other immune-mediated diseases. This study identifies a subset of uveitis patients distinguished by expression of a specific pANCA marker antibody. The characteristics of this antibody are similar to the pANCA antibody present in most patients with ulcerative colitis. Expression of the pANCA autoantibody in uveitis patients is a susceptibility marker for other immune-mediated diseases.

Adult↗

Phage display cloning and characterization of an immunogenetic marker (perinuclear anti-neutrophil cytoplasmic antibody) in ulcerative colitis.

Ulcerative colitis (UC) is genetically associated with a marker serum Ab (pANCA), identified by its reactivity with a neutrophil Ag. This study utilized phage display technology to clone and characterize pANCA, which has resisted conventional isolation strategies. Since spontaneous pANCA-secreting B cells are detectable in UC lamina propria lymphocytes, this cell source was used to construct a complete IgG1-kappa Ig library. Selection of phage by panning with fixed neutrophils yielded a 195-fold enrichment after five cycles of panning. BstN1 fingerprinting of the enriched library revealed two predominant clones, and DNA sequencing demonstrated highly homologous heavy and light chain variable region segments. Clones were reengineered to express soluble Fab, and neutrophil binding was verified by ELISA. Detailed studies with the two recombinant Fabs, NANUC-1 and -2, validated their identity with serum pANCAs by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. NANUC-1 and -2, like serum UC-pANCA, lack reactivity with previously characterized ANCA-reactive neutrophil proteins and thus detect a novel Ag(s). This study demonstrates the feasibility of selecting phage display Ab libraries on uncharacterized biologic substrates to isolate marker Abs of pathogenic importance.

Amino Acid Sequence↗

Expression of a novel autoantibody defined by the VH3-15 gene in inflammatory bowel disease and Campylobacter jejuni enterocolitis.

This study newly introduces anti-VH mAbs to assess the role of clonal B cell activity in inflammatory bowel disease. Immunohistochemistry of colonic biopsies in ulcerative colitis (UC) and Crohn's disease (CD), but not unaffected individuals, demonstrated uniform staining of intravascular erythrocytes with BK2, a monoclonal specific for the VH3-15 Ig heavy chain gene product. Staining was caused by erythrocytes opsinized in vivo by anti-erythrocyte Abs present in patient sera and by using the VH3-15 gene product. The erythrocyte Ag was identified by immunoprecipitation as 22- and 28-kDa membrane proteins. A direct flow cytometric assay was developed to measure this serum autoantibody and was tested in 101 individuals with UC, CD, other acute or chronic colitis, and healthy controls. Compared with normal subjects, BK2+ anti-erythrocyte Abs were elevated in most sera from patients with CD and UC (including postcolectomy). BK2+ anti-erythrocyte Abs also were elevated in 10 of 38 noninflammatory bowel disease patients, all of whom had Campylobacter jejuni enterocolitis. These findings suggest that a common immunopathogenetic factor, manifested by VH3-15 B cell activation may be shared in UC, CD, and Campylobacter jejuni enterocolitis.

Amino Acid Sequence↗

Simultaneous administration of Haemophilus influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine with routine diphtheria-tetanus-pertussis and inactivated poliovirus vaccinations of childhood.

A Haemophilus influenzae type b capsular polysaccharide-diphtheria toxoid conjugate vaccine (PRP-D) is capable of protecting infants against invasive H. influenzae diseases. Therefore it is very likely that it will be incorporated in routine vaccination schedules during the next few years. In order to test the suitability of simultaneous administration of PRP-D and other vaccines we administered it to 25 infants mixed with diphtheria-tetanus-pertussis vaccine at 3, 4 and 6 months and simultaneously, but in a separate syringe, with inactivated polio vaccine at 12 months. A comparison group of equal size received only diphtheria-tetanus-pertussis and inactivated poliovirus vaccines. The concentration of postvaccination antibodies to diphtheria toxoid was 0.411 IU/ml in the group that received PRP-D vs. 0.352 IU/ml in the comparison group, to tetanus toxoid 3.666 vs. 3.668 IU/ml and the neutralization titer to poliovirus type 1 was 370 vs. 320 units in the comparison group, to type 2 titer values were 230 vs. 270 units and to type 3, respectively, 210 vs. 290 units. Thus the seroresponse to antigens in routine vaccines was not affected by the presence of PRP-D in the vaccination schedule, and PRP-D can safely and effectively be included in the vaccination schedule of infancy.

Bacterial Vaccines↗

Variable quantitation of Haemophilus influenzae type b anticapsular antibody by radioantigen binding assay.

The measurement of antibody to Haemophilus influenzae type b capsular polysaccharide is important in the study of natural immunity and in the immunogenicity evaluation of H. influenzae type b vaccines. Several radioantigen binding assays (RABA) have been developed to measure H. influenzae type b anticapsular antibody, but recent immunogenicity data obtained with structurally similar vaccines suggest major differences in antibody quantitation in different laboratories. To evaluate interlaboratory variability in the measurement of anticapsular antibody levels, we blindly evaluated a sample of 40 pre- and postimmunization sera by eight RABAs in different laboratories. Evaluation of RABA methods revealed differences in polysaccharide antigens, radiolabeling methods, concentration and volume of antigen and antibody, and other assay methods. The reported results of assays varied significantly between laboratories (up to sixfold differences in geometric means), in part because of differences in assay sensitivity and different proportions of samples having undetectable levels of antibody (0 to 65% of specimens with undetectable levels). After standardizing the limit of sensitivity for all assays (0.125 microgram/ml), the results of all combinations of paired analyses of RABA assays correlated well (r = 0.88 to 0.99) but the geometric mean levels still varied as much as twofold. For individual sera, the differences between paired assays often were substantial (P less than or equal to 0.0001, paired t test), with some results varying as much as 64-fold. Differences were greatest for lower levels of antibody. There was good comparability and interlaboratory reproducibility of some assays but not of others. Intrinsic or extrinsic labeling of the antigen was not a major determinant of comparability. In most instances, the current variation in the quantitation of antibody levels by these assays precludes interassay comparisons. A standardized measurement of antibody needs to be developed to adequately compare results between different H. influenzae type b immunogenicity studies.

Antibodies, Bacterial↗

Immunogenicity in infants of a vaccine composed of Haemophilus influenzae type b capsular polysaccharide mixed with DPT or conjugated to diphtheria toxoid.

Fifty-three healthy infants received either Haemophilus influenzae type b capsular polysaccharide (PRP) mixed with diphtheria-pertussis-tetanus vaccine (DPT) or PRP conjugated covalently to diphtheria toxoid (PRP-D). The immunizations were given at 3, 5, 7, and either 14 or 18 months of age and were well tolerated. The geometric mean titers of antibody to PRP at eight months of age (after the first immunizations) were 0.26 micrograms/ml in the PRP + DPT group and 1.56 micrograms/ml in the PRP-D group. After the fourth dose, an IgG response was seen in both groups. The PRP + DPT group had a geometric mean level of antibody to PRP of 3.98 micrograms/ml at 19 months and the PRP-D group, 31.22 or 24.00 micrograms/ml at 15 or 19 months, respectively. Compared with previously published data of children immunized with one dose of PRP at 18 months, the mean level of antibody to PRP in the PRP-D group at 19 months was significantly (P less than .001) higher, whereas that in the PRP + DPT group was not.

Antibodies, Bacterial↗