- Email: [email protected]
Phage display cloning and characterization of the ulcerative colitis specific perinuclear anti-neutrophil cytoplasmic antibody (pANCA)
April 1 9 9 5
•
PHAGE DISPLAY CLONING AND CHARACTERIZATION OF THE ULCERATIVE COLITIS SPECIFIC PERINUCLEAR ANTINEUTROPHIL CYTOPLASMIC ANTIBODY (pANCA). M.P. E~r~ena*, S.R. Targan¶, A. Vidrich¶, J. Braun*. Department of Pathology .and Labgrat0ry Medicine*, and Medicine¶, and Molecular Biology Institute*, UCLA, Los Angeles, CA and ¶Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, Los Angeles, CA Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) are present in the sera of most patients with ulcerative colitis (UC) and distinguish genetically disparate UC and Crohn's disease subsets. Serum pANCA is a familial trait associated with disease susceptibility and disease associated MHC haplotypes. The UC-associated pANCA is distinguished from other ANCAs (e.g., in Wegener's granulomatosis) by its distinct immunocytochemical staining pattern, localization to the subnuclear membrane, and DNase 1 sensitivity (i.e. disappearancc of pANCA immunofluorescence following treatment of rlcutrophils with DNase 1). pANCA and its target antigen have remained uncharacterized because of its resistance to c0nventiona! cloning strategies. This study utilizes phage display technology to clone and characterize UC-pANCA. Phage display technology allows the construction and display of large immunogtobulin Fab libraries (108) on the surface Of M]3 filamentous phage. Antibodies of desired specificity can be selected from the library by bioparming. Since spontaneous pANCA-secreting B cells are detectable in UC lamina propria lymphocytes (Gastro. I06:A781, 1994), this cell source was used to construct a complete IgGl-kappa immunoglobulin library. This library was selected for pANCA reactivity by panning with fixed neutrophils and 192 fold enrichment was achieved after 5 panning cycles. BstN1 fingerprinting of the enriched library revealed two predominant selected clones, and DNA sequencing demonstrated highly homologous heavy and light chain vari~hlc region segments suggesting that serum pANCA may be oligoclonal. Clones were rccngincered to express soluble Fab and neutrophi! binding was verified by ELISA. Detailed studies with one Fab, 5-3, validated its identity with the serum pANCA of the patient from which the library was derived by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. Like serum UC~-pANCA, Fab 5-3 lacks reactivity with previously characterized pANCA-reacrive neutrophil proteins and thus detects a novel antigen. We are in the process of using 5-3 to clone and characterize the neutrophil antigen. Characterization of disease specific marker antibodies such as pANCA are important in diagnosis and in the seurch for candidate antigenic targets that may be responsible for the pathogenesis of disease.
Immunology, Microbiology, and Inflammatory Disorders
A815
PERIANALCONDYLOMAACUMINATA: HIV-POSITIVEVS. HIV-NEGATIVE PATIENTS. W.R.Ehrmantraut.Jr., B.A.Orkin, LE.Smith. The George Washington University, Washington, DC Perianal condylomata acuminata is a troublesome and common disease causeu oy the human papilloma virus. HIV seropositive (HI',/+) patients commonly have perianal condylomata and they may be difficult to treat. Our hypothesis was that HIV+ patients present with more severe disease, have more extensive recurrence and take longer to render disease free than HIV seronegative (HIV-) oatients. We reviewed all patients presenting to the Division of Colon and Rectal Surgery between the years of 1985 and 1994 with a diagnosis of perianal condyloma acuminata. All patients were treated using a standard protocol with the goal of complete initial destruction of visiblelesions using electmcoagulation and/or CO2 laser fulguration. Chemical treatments were occasionally used in the early years of the study but were later abandoned. Patients were closely followed at 3 to 6 week intervals until disease free for one year. Seventy of disease at presentation and at recurrence was graded on the basis of involved surface area and number of lesions. Patients with Grade 1 disease (mild) had less than or equal to 10 scattered small lesions, patients with Grade 2 disease (moderate] ane 11 to 20 lesions, and patients with Grade 3 disease (severe) had greater than 20 lesions and/or large confluent involved areas Data were analyzed by severity at presentation, by time to first recurrence, by severity at first recurrence and by number of treatment until rendered persistently disease free. Recurrence data was corrected for severity of disease at presentation. Condyloma acuminata were identified in 122 ;~atients presenting during this 9 year period. 55 were HIV+. 40 were HtV-. and 27 were of unknown HIV status. The following results compare the HIV+ and HIV- patients only. Vadable HIV+(55) HIV- (40) P value Severity at presentation* 2.2+0.7 1,5+0.6 .0001 Follow up (mons) 18.9 15.3 N.S.D No. follow up visits 6.1+5.6 6.6+6.5 N.S.D, Severity at recurrence" 1.5+0.5 1.1+0.3 .0004 No. oositive follow up visits 5,2+4.7 4.9+4.8 NS.D - Severity scale Grades 1-3. Follow up was e0uivalent in both patient groups. Stepwise regression analysis eliminated sexual preference, gender, age and race as factors identifying HIV status as the only significant variable. HIV+ patients had more extensive disease at initial presentation and at recurrence. Of those patients with recurrence, there Was no difference in the number of visits with recurrence. This data support the hypothesis that HIV-seropositive patients have more extensive anal condylomata acuminata both on initial presentation and on follow up.
• PATTERN OF G A S T R O I N T E S T I N A L SYMPTOMS ACROSS THE MENSTRUAL CYCLE IN INFLAMMATORY BOWEL DISEASE PATIENTS. D. Eilerina, J. O'Brien, A. Pittler, C. Randall. W a s h i n g t o n U n i v e r s i t y School of Medicine, St. Louis, Missouri.
• ANTI-CD4-ANTIBODY TREATMENT IN INFLAMMATORY BOWEL DISEASE WITHOUT A LONG CD4+-CELL DEPLETION. J. Emmrich. M.Seyfarth, S.Liebe, F.Emmrich. Dept. of Medicine, University of Restock, Institutes of Immunology, Universities of Litheck and Leipzig, Germany
Clinical observations have suggested an increase in g a s t r o i n t e s t i n a l (GI) s y m p t o m a t o l o g y in m e n s t r u a t i n g w o m e n w i t h Inflammatory Bowel Disease (IBD) during the p r e - m e n s e s and m e n s e s phases of the menstrual cycle. T h i r t e e n p r e - m e n o p a u s a l p a t i e n t s (pts) w i t h IBD (range: 27 to 46 yrs) p a r t i c i p a t e d in a p r o s p e c t i v e study to d e t e r m i n e the p a t t e r n of GI symptoms across the m e n s t r u a l cycle. All pts had c o n f i r m e d IBD (three: u l c e r a t i v e colitis; ten: Crohn's disease) according to c o n v e n t i o n a l l y a c c e p t e d radiologic, h i s t o l o g i c and e n d o s c o p i c criteria. The study p r o t o c o l included a b a s e l i n e interview, an initial q u e s t i o n n a i r e including GI and g y n e c o l o g i c history, a couple clinic v i s i t s and a daily d i a r y recording GI symptoms and o t h e r features. The d u r a t i o n of o b s e r v a t i o n per pt involved a m i n i m u m of two menstrual cycles. The pts were also instructed to call the study c o o r d i n a t o r if they had any questions about c o m p l e t i n g t h e i r diary. A modified HarveyB r a d s h a w (H-B) IBD a c t i v i t y index was implemented to d o c u m e n t clinical d i s e a s e activity. In a d d i t i o n to the i n f o r m a t i o n n e e d e d to c o m p l e t e H-B scores, data was also c o m p i l e d on a c t i v i t i e s of daily living. Results: 92% (12/13) of the women noted an increase in s y m p t o m s at menses, 62% (8/13) r e p o r t e d an increase at pre-menses. Increases in the HarveyB r a d s h a w scores w e r e also noted during the premenses and m e n s e s p h a s e s of the menstrual cycle. The m o s t c o m m o n l y r e p o r t e d symptoms were diarrhea, abdominal cramping, fatigue and arthritic pain. O t h e r s y m p t o m s i n c l u d e d nausea, eye irritation, g a s e o u s n e s s and headache. Conclusion: P r e m e n o p a u s a l w o m e n w i t h IBD r e p o r t e d a m p l i f i e d GI symptoms at the p r e - m e n s e s and menses p h a s e s of the m e n s t r u a l cycle.
Baehizround: Previously we have reported on the first administration of mouse monoclonal anti-CD4 antibodies (MAX.16H5) to treat patients with chronic inflammatory bowel diseases (Lancet 338:570,1991). Clinical improvement after antibody treatment has also been documented by DEUSCH et al. (Gastroenterology 104:A691,1993) using the chimeric monoclonal anti-CD4 antibody cM-T412 but with a long lasting significant drop in circulating CD4+-cells. The anti-CD4 antibody 16H5 did not involve such an immtmosuppreesion and we therefore continued our study. Methods: We studied 12 patients with severe, chronic active ulcerative colitis (UC, n=7) or Crolm's disease (CD, u=3). In an open clinical trial, mouocloml antibody 16H5 was infused daily at a dosage of 0.3 mg/kg body weight for 7 consecutive days. Concommittant drug therapy was prednisolone not more than 10rag daily and mesalazine. Two patients with UC and two patients with CD got a second therapy cycle four weeks after the first. For evaluation of the clinical response, we used the Crohn's disease activity index (CDAD and the clinical activity index (CAD according RACHM][LEWITZ for UC. Laboratory parameters indinating inflammatory activity (ESR, WBC, CRP, orosomucoids) were also measured, Lymphocyte subpopalations, anti- mouse antibodies (HAMA) and lymphocyte stimulation by mitogens were characterized. Results: We reached a clinical remission in three patients suffering for CD over four to sixteen weeks. Remission was achieved in four patients with UC for a time period between four and eight weeks. One patient came into a complete remission lasting for three years. In two patients there was no effect of our therapy on the activity of the disease. The other four patients, who were treated with two cymes had a remisson over 5 to 8 months. In the patients with remission we could show a reduction in the levels of acute-phase parameters, as demonSxated by ESR, CRP, WBC as well as orosomucoid. Under the antibody infusion the CD4 cell count droped quickly but 24 hours later the CD4 cell counts were nearly normal. HAMA codd be detected already two weeks inter. The other lymphocyte subpopulations were not a.fleeted by the therapy. None of our patients developed side-effects from antibody treatment. Conclusions: Aati-CD4 antibody treatment is effective in inducing remission in inflammatory bowel disease when conventional drugs have failed. After two therapy cycles the duration of remission was longer than using a single cycle. A prolonged immunosuppression did not occur. Supported by the BMFT, FKZ 01/ZU/$607.
•
PHAGE DISPLAY CLONING AND CHARACTERIZATION OF THE ULCERATIVE COLITIS SPECIFIC PERINUCLEAR ANTINEUTROPHIL CYTOPLASMIC ANTIBODY (pANCA). M.P. E~r~ena*, S.R. Targan¶, A. Vidrich¶, J. Braun*. Department of Pathology .and Labgrat0ry Medicine*, and Medicine¶, and Molecular Biology Institute*, UCLA, Los Angeles, CA and ¶Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, Los Angeles, CA Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) are present in the sera of most patients with ulcerative colitis (UC) and distinguish genetically disparate UC and Crohn's disease subsets. Serum pANCA is a familial trait associated with disease susceptibility and disease associated MHC haplotypes. The UC-associated pANCA is distinguished from other ANCAs (e.g., in Wegener's granulomatosis) by its distinct immunocytochemical staining pattern, localization to the subnuclear membrane, and DNase 1 sensitivity (i.e. disappearancc of pANCA immunofluorescence following treatment of rlcutrophils with DNase 1). pANCA and its target antigen have remained uncharacterized because of its resistance to c0nventiona! cloning strategies. This study utilizes phage display technology to clone and characterize UC-pANCA. Phage display technology allows the construction and display of large immunogtobulin Fab libraries (108) on the surface Of M]3 filamentous phage. Antibodies of desired specificity can be selected from the library by bioparming. Since spontaneous pANCA-secreting B cells are detectable in UC lamina propria lymphocytes (Gastro. I06:A781, 1994), this cell source was used to construct a complete IgGl-kappa immunoglobulin library. This library was selected for pANCA reactivity by panning with fixed neutrophils and 192 fold enrichment was achieved after 5 panning cycles. BstN1 fingerprinting of the enriched library revealed two predominant selected clones, and DNA sequencing demonstrated highly homologous heavy and light chain vari~hlc region segments suggesting that serum pANCA may be oligoclonal. Clones were rccngincered to express soluble Fab and neutrophi! binding was verified by ELISA. Detailed studies with one Fab, 5-3, validated its identity with the serum pANCA of the patient from which the library was derived by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. Like serum UC~-pANCA, Fab 5-3 lacks reactivity with previously characterized pANCA-reacrive neutrophil proteins and thus detects a novel antigen. We are in the process of using 5-3 to clone and characterize the neutrophil antigen. Characterization of disease specific marker antibodies such as pANCA are important in diagnosis and in the seurch for candidate antigenic targets that may be responsible for the pathogenesis of disease.
Immunology, Microbiology, and Inflammatory Disorders
A815
PERIANALCONDYLOMAACUMINATA: HIV-POSITIVEVS. HIV-NEGATIVE PATIENTS. W.R.Ehrmantraut.Jr., B.A.Orkin, LE.Smith. The George Washington University, Washington, DC Perianal condylomata acuminata is a troublesome and common disease causeu oy the human papilloma virus. HIV seropositive (HI',/+) patients commonly have perianal condylomata and they may be difficult to treat. Our hypothesis was that HIV+ patients present with more severe disease, have more extensive recurrence and take longer to render disease free than HIV seronegative (HIV-) oatients. We reviewed all patients presenting to the Division of Colon and Rectal Surgery between the years of 1985 and 1994 with a diagnosis of perianal condyloma acuminata. All patients were treated using a standard protocol with the goal of complete initial destruction of visiblelesions using electmcoagulation and/or CO2 laser fulguration. Chemical treatments were occasionally used in the early years of the study but were later abandoned. Patients were closely followed at 3 to 6 week intervals until disease free for one year. Seventy of disease at presentation and at recurrence was graded on the basis of involved surface area and number of lesions. Patients with Grade 1 disease (mild) had less than or equal to 10 scattered small lesions, patients with Grade 2 disease (moderate] ane 11 to 20 lesions, and patients with Grade 3 disease (severe) had greater than 20 lesions and/or large confluent involved areas Data were analyzed by severity at presentation, by time to first recurrence, by severity at first recurrence and by number of treatment until rendered persistently disease free. Recurrence data was corrected for severity of disease at presentation. Condyloma acuminata were identified in 122 ;~atients presenting during this 9 year period. 55 were HIV+. 40 were HtV-. and 27 were of unknown HIV status. The following results compare the HIV+ and HIV- patients only. Vadable HIV+(55) HIV- (40) P value Severity at presentation* 2.2+0.7 1,5+0.6 .0001 Follow up (mons) 18.9 15.3 N.S.D No. follow up visits 6.1+5.6 6.6+6.5 N.S.D, Severity at recurrence" 1.5+0.5 1.1+0.3 .0004 No. oositive follow up visits 5,2+4.7 4.9+4.8 NS.D - Severity scale Grades 1-3. Follow up was e0uivalent in both patient groups. Stepwise regression analysis eliminated sexual preference, gender, age and race as factors identifying HIV status as the only significant variable. HIV+ patients had more extensive disease at initial presentation and at recurrence. Of those patients with recurrence, there Was no difference in the number of visits with recurrence. This data support the hypothesis that HIV-seropositive patients have more extensive anal condylomata acuminata both on initial presentation and on follow up.
• PATTERN OF G A S T R O I N T E S T I N A L SYMPTOMS ACROSS THE MENSTRUAL CYCLE IN INFLAMMATORY BOWEL DISEASE PATIENTS. D. Eilerina, J. O'Brien, A. Pittler, C. Randall. W a s h i n g t o n U n i v e r s i t y School of Medicine, St. Louis, Missouri.
• ANTI-CD4-ANTIBODY TREATMENT IN INFLAMMATORY BOWEL DISEASE WITHOUT A LONG CD4+-CELL DEPLETION. J. Emmrich. M.Seyfarth, S.Liebe, F.Emmrich. Dept. of Medicine, University of Restock, Institutes of Immunology, Universities of Litheck and Leipzig, Germany
Clinical observations have suggested an increase in g a s t r o i n t e s t i n a l (GI) s y m p t o m a t o l o g y in m e n s t r u a t i n g w o m e n w i t h Inflammatory Bowel Disease (IBD) during the p r e - m e n s e s and m e n s e s phases of the menstrual cycle. T h i r t e e n p r e - m e n o p a u s a l p a t i e n t s (pts) w i t h IBD (range: 27 to 46 yrs) p a r t i c i p a t e d in a p r o s p e c t i v e study to d e t e r m i n e the p a t t e r n of GI symptoms across the m e n s t r u a l cycle. All pts had c o n f i r m e d IBD (three: u l c e r a t i v e colitis; ten: Crohn's disease) according to c o n v e n t i o n a l l y a c c e p t e d radiologic, h i s t o l o g i c and e n d o s c o p i c criteria. The study p r o t o c o l included a b a s e l i n e interview, an initial q u e s t i o n n a i r e including GI and g y n e c o l o g i c history, a couple clinic v i s i t s and a daily d i a r y recording GI symptoms and o t h e r features. The d u r a t i o n of o b s e r v a t i o n per pt involved a m i n i m u m of two menstrual cycles. The pts were also instructed to call the study c o o r d i n a t o r if they had any questions about c o m p l e t i n g t h e i r diary. A modified HarveyB r a d s h a w (H-B) IBD a c t i v i t y index was implemented to d o c u m e n t clinical d i s e a s e activity. In a d d i t i o n to the i n f o r m a t i o n n e e d e d to c o m p l e t e H-B scores, data was also c o m p i l e d on a c t i v i t i e s of daily living. Results: 92% (12/13) of the women noted an increase in s y m p t o m s at menses, 62% (8/13) r e p o r t e d an increase at pre-menses. Increases in the HarveyB r a d s h a w scores w e r e also noted during the premenses and m e n s e s p h a s e s of the menstrual cycle. The m o s t c o m m o n l y r e p o r t e d symptoms were diarrhea, abdominal cramping, fatigue and arthritic pain. O t h e r s y m p t o m s i n c l u d e d nausea, eye irritation, g a s e o u s n e s s and headache. Conclusion: P r e m e n o p a u s a l w o m e n w i t h IBD r e p o r t e d a m p l i f i e d GI symptoms at the p r e - m e n s e s and menses p h a s e s of the m e n s t r u a l cycle.
Baehizround: Previously we have reported on the first administration of mouse monoclonal anti-CD4 antibodies (MAX.16H5) to treat patients with chronic inflammatory bowel diseases (Lancet 338:570,1991). Clinical improvement after antibody treatment has also been documented by DEUSCH et al. (Gastroenterology 104:A691,1993) using the chimeric monoclonal anti-CD4 antibody cM-T412 but with a long lasting significant drop in circulating CD4+-cells. The anti-CD4 antibody 16H5 did not involve such an immtmosuppreesion and we therefore continued our study. Methods: We studied 12 patients with severe, chronic active ulcerative colitis (UC, n=7) or Crolm's disease (CD, u=3). In an open clinical trial, mouocloml antibody 16H5 was infused daily at a dosage of 0.3 mg/kg body weight for 7 consecutive days. Concommittant drug therapy was prednisolone not more than 10rag daily and mesalazine. Two patients with UC and two patients with CD got a second therapy cycle four weeks after the first. For evaluation of the clinical response, we used the Crohn's disease activity index (CDAD and the clinical activity index (CAD according RACHM][LEWITZ for UC. Laboratory parameters indinating inflammatory activity (ESR, WBC, CRP, orosomucoids) were also measured, Lymphocyte subpopalations, anti- mouse antibodies (HAMA) and lymphocyte stimulation by mitogens were characterized. Results: We reached a clinical remission in three patients suffering for CD over four to sixteen weeks. Remission was achieved in four patients with UC for a time period between four and eight weeks. One patient came into a complete remission lasting for three years. In two patients there was no effect of our therapy on the activity of the disease. The other four patients, who were treated with two cymes had a remisson over 5 to 8 months. In the patients with remission we could show a reduction in the levels of acute-phase parameters, as demonSxated by ESR, CRP, WBC as well as orosomucoid. Under the antibody infusion the CD4 cell count droped quickly but 24 hours later the CD4 cell counts were nearly normal. HAMA codd be detected already two weeks inter. The other lymphocyte subpopulations were not a.fleeted by the therapy. None of our patients developed side-effects from antibody treatment. Conclusions: Aati-CD4 antibody treatment is effective in inducing remission in inflammatory bowel disease when conventional drugs have failed. After two therapy cycles the duration of remission was longer than using a single cycle. A prolonged immunosuppression did not occur. Supported by the BMFT, FKZ 01/ZU/$607.