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H A Homburger

Publications and source records attributed to H A Homburger.

At least 19 recordsLinked to original sources
PubMed1998

Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes.

Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids.

Animals↗
PubMed1998

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide.

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and delta-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, delta-rPR3-S176A does not. Culture supernatants of rPR3-S176A and delta-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With delta-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1998

Detection of antinuclear antibodies: comparative evaluation of enzyme immunoassay and indirect immunofluorescence methods.

OBJECTIVE: To evaluate a commercial microtiter enzyme immunoassay for antinuclear antibodies (ANA) by comparing the results of tests performed with this assay to an established indirect immunofluorescence method performed on human epitheliod cell substrate slides. DESIGN: Both analytical methods were used to test for the presence and levels of ANA in stored sera from 313 patients previously shown to have detectable ANA and from 102 healthy control subjects. Follow-up tests for specific autoantibodies (anti-dsDNA antibodies and antibodies to extractable nuclear antigens [ENA]) were performed on all sera from patients. The medical histories of all patients were reviewed to determine the presence of systemic rheumatic diseases (SRDs). Different cut-off levels of positivity were examined to determine the sensitivity and predictive values of positive results on the enzyme immunoassay for detecting patients with SRDs or sera with positive tests for specific autoantibodies. RESULTS: Among patients with clinically diagnosed SRDs (n = 197), the enzyme immunoassay was positive for ANA (> or =1 U) in 100% and the indirect immunofluorescence method was positive (titer > or =40) in 95.4% of cases. Among ANA-positive patients with no SRDs (n = 116), testing by enzyme immunoassay and indirect immunofluorescence yielded positive results in 97.6% and 75.6% of cases, respectively. Among healthy control subjects, each of the two methods was positive in 15% of cases. As expected, most patients with SRDs had higher levels of ANA than did ANA-positive patients with other clinical diagnoses. A cut-off level of > or =3 U on the enzyme immunoassay correctly classified 77% of patients with a SRD as "positive" and 88% of patients with other clinical diagnoses as "negative." The probability of detecting a positive result for specific autoantibodies on second-order testing increased directly with the level of ANA. A cut-off level of > or =3 U had a sensitivity of 92% for identifying sera with positive specific autoantibodies, and results > or =3 U had a predictive value of 52% for a positive second-order test result. CONCLUSION: Enzyme immunoassay is substantially equivalent to indirect immunofluorescence for detecting clinically important ANA. Cut-off levels for positive results on the enzyme immunoassay can be established that optimize the usefulness of this method in diagnostic algorithms for specific autoantibodies.

Antibodies, Antinuclear↗
PubMed1997

Quantitative measurement of autoantibodies to recombinant mitochondrial antigens in patients with primary biliary cirrhosis: relationship of levels of autoantibodies to disease progression.

We examined the clinical usefulness of measurements of antimitochondrial autoantibodies (AMA) in predicting disease progression in patients with primary biliary cirrhosis (PBC). We determined the relationships between AMA levels measured by indirect immunofluorescence (IF) and those measured by quantitative enzyme immunoassays (EIAs) using recombinant 2-oxo-acid dehydrogenase complex (2-OADC) proteins and the Mayo Risk Score, an established indicator of disease progression in primary biliary cirrhosis (PBC). Results of tests for AMA by either method correlated weakly (r = .24 to .30) with disease progression as indicated by Mayo Risk Scores. The levels of AMA to 2-OADC proteins varied by more than 200-fold between patients but remained relatively constant over time in individual patients. Despite being positively correlated with Mayo Risk Score results, the levels of AMA to 2-OADC proteins were not useful for predicting disease progression in individual patients with PBC. In addition, we found no significant differences in the levels of autoantibodies to 2-OADC proteins among patients with different histological stages of disease. Our results show that measurements of AMA by IF or by quantitative EIA methods with recombinant 2-OADC proteins are not useful parameters for predicting disease progression in patients with PBC.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1997

Does antimitochondrial antibody status affect response to treatment in patients with primary biliary cirrhosis? Outcomes of ursodeoxycholic acid therapy and liver transplantation.

Approximately 5% to 10% of patients with features otherwise consistent with primary biliary cirrhosis (PBC) lack antimitochondrial antibodies (AMA). Most of these patients have other autoantibodies, a syndrome recently named "autoimmune cholangitis." We report our experience in patients with AMA-negative PBC treated with ursodeoxycholic acid (UDCA) and/or liver transplantation (OLT). The study of response to UDCA was performed as follows. While recruiting patients for a previously reported multicenter trial, we identified 8 patients with AMA-negative PBC. The patients were given UDCA and followed up at regular intervals. The characteristics of AMA-negative patients at presentation were similar to those of AMA-positive patients with PBC. The clinical outcomes and sequential liver biochemistries of UDCA treatment were also comparable with those of AMA-positive patients. The study of outcome of OLT was performed as follows. We identified OLT recipients at the Mayo Clinic who had clinical, radiological, and histological features compatible with PBC. Their pretransplant AMA status was determined, and each AMA-negative patient was paired with 2 AMA-positive patients. Of 85 OLT recipients with a diagnosis of PBC, 6 (7.1%) were AMA negative, including 1 who had undergone UDCA therapy. After a median of 36 months of follow-up, graft and patient survival rates and subsequent histological changes (disease recurrence and steroid-resistant or late rejections) were comparable in AMA-negative and -positive PBC patients. In summary, in our experience of 13 AMA-negative PBC patients (including 9 who met the criteria for a diagnosis of autoimmune cholangitis), treatment with UDCA or OLT resulted in similar outcomes to those found in AMA-positive patients. We conclude that AMA status does not affect the response in PBC patients to treatment with UDCA or OLT.

Adult↗
PubMed1997

Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA).

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjogren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P < 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1997

Peripheral neuropathy associated with sicca complex.

Peripheral neuropathy occurs in Sjögren's syndrome, a disorder in which systemic immunologic phenomena, including vasculitis, are common. Neuropathy also occurs with isolated sicca complex (keratoconjunctivits sicca and xerostomia); whether this represents a distinct syndrome is unclear. We retrospectively studied 54 patients with sicca complex and peripheral neuropathy to determine mode of presentation, neuropathic patterns, frequency and pattern of serologic abnormalities, and frequency of systemic disease, including necrotizing vasculitis. Peripheral neuropathy was the presenting problem in 87%. Although sicca symptoms occurred in 93%, they were a presenting complaint in only 11% and were usually mild, reported only after specific inquiry. Minor salivary gland biopsy was positive in 73%. Sensory neuropathies strongly predominated; 61% of patients manifested either sensory polyneuropathy or polyganglionopathy. Less common patterns included sensorimotor polyneuropathy (17%) and polyradiculoneuropathy (11%). Vasculitic neuropathy was demonstrated in only two patients, but nonspecific epineurial inflammation was present in 70% of nerve biopsies. Clinical evidence of systemic disease was uncommon, particularly in the sensory polyganglionopathy group, in whom extraglandular features other than weight loss occurred in only 1 of 12 patients. Antibodies to extractable nuclear antigens, the most specific serologic marker of Sjögren's syndrome, were present in 10.4%. We conclude that peripheral neuropathy and isolated sicca complex form a distinctive syndrome in which neuropathy is the presenting feature and sicca is easily overlooked; sensory polyneuropathy and polyganglionopathy predominate; serology is confirmatory but very insensitive; and extraglandular disease, including vasculitis, is uncommon compared with typical Sjögren's syndrome. Tests of ocular or salivary involvement are needed for diagnosis, and demonstration of inflammation in biopsied nerve is supportive. Improved definition of this disorder should permit further studies of natural history and efficacy of immunotherapy.

Adult↗
PubMed1997

Octreotide inhibition of flushing and colonic motor dysfunction in carcinoid syndrome.

OBJECTIVES: Previous studies showed increased plasma motilin and substance P concentrations and accelerated motor function in the small bowel and colon in patients with carcinoid diarrhea. Octreotide is beneficial in patients with carcinoid syndrome. Our hypothesis was that octreotide inhibits accelerated motility and gut neuropeptides in carcinoid syndrome. METHODS: In 12 patients with metastatic carcinoid syndrome, we investigated the effect of octreotide 50 microg s.c. t.i.d (n = 6) or placebo (n = 6) on postprandial symptoms, GI transit, colonic motility, and circulating levels of selected circulating peptides and amines. RESULTS: Octreotide reduced postprandial flushing (p = 0.03) but not pain. Octreotide significantly retarded overall colonic transit and proximal colonic emptying (p < 0.05); it tended to prolong small bowel transit time (p = 0.13) and to reduce postprandial colonic tone (p = 0.08) compared with placebo. Octreotide also reduced circulating levels of peptide YY, neurotensin, vasoactive intestinal polypeptide, and substance P but had no effect on plasma motilin, neuropeptide Y, calcitonin gene-related peptide, or histamine after meal ingestion. CONCLUSION: Octreotide ameliorates gut motor dysfunctions that characterize carcinoid diarrhea; the potential role of specific antagonism of serotonin, substance P, and vasoactive intestinal polypeptide alone or in combination with agents that inhibit their release in carcinoid diarrhea deserves further study.

Aged↗
PubMed1994

Lesions of the respiratory tract associated with the finding of anti-neutrophil cytoplasmic autoantibodies with a perinuclear staining pattern.

OBJECTIVE: To characterize the clinicopathologic spectrum of respiratory tract involvement in patients with positive results of immunofluorescence microscopy for anti-neutrophil cytoplasmic autoantibodies with a perinuclear staining pattern (p-ANCA) and to assess the clinical value of p-ANCA testing. DESIGN: We retrospectively reviewed the medical records of all patients at Mayo Clinic Rochester in whom p-ANCA were detected by indirect immunofluorescence microscopy during 1992. MATERIAL AND METHODS: Additional target antigen identification was performed with use of enzyme-linked immunosorbent assays for antibodies against myeloperoxidase (MPO) and proteinase 3. We summarized the clinical findings in MPO-positive and MPO-negative patients. RESULTS: Sera were positive for p-ANCA in 42 of 2,381 patients (1.8%). In 13 of these 42 patients (31%), the respiratory tract was involved. Twelve patients had chest roentgenographic abnormalities, including a diffuse alveolar filling pattern (N = 8), a diffuse interstitial pattern (N = 2), and a combined interstitial and alveolar pattern (N = 2); three others had nasal inflammation. Ten of 16 sera tested were positive for MPO, and proteinase 3 antibodies were detected in 1 specimen. All patients with alveolar hemorrhage were positive for MPO antibodies. CONCLUSION: Testing for p-ANCA by immunofluorescence microscopy discloses a wide range of clinical disorders distinct from the main cytoplasmic-staining ANCA-associated disease--namely, Wegener's granulomatosis. In particular, the respiratory tract is affected much less frequently. Further evaluation of positive p-ANCA immunofluorescence test results by enzyme-linked immunosorbent assay to determine whether MPO is the target antigen is necessary to obtain clinically useful information from this test.

Adolescent↗
PubMed1994

Soluble T lymphocyte markers in the diagnosis of cellular rejection and cytomegalovirus hepatitis in liver transplant recipients.

Hepatic dysfunction following liver transplantation is often caused by cellular rejection or infection with cytomegalovirus. These etiologies can at times be difficult to differentiate. We measured the levels of soluble T lymphocyte markers sIL2R, sCD4, and sCD8 in serum as possible diagnostic indicators of cellular rejection and cytomegalovirus hepatitis. Pretransplant levels, and serial post-transplant levels of soluble T lymphocyte markers were measured in five control patients without cellular rejection or cytomegalovirus infection, ten patients with cellular rejection, and six patients with cytomegalovirus hepatitis. In all cases, cellular rejection and cytomegalovirus hepatitis were documented with liver histology. For each group of patients, we calculated the mean ratio of the post-transplant marker level divided by the pre-transplant level. We found an elevation in the mean ratio of sIL2R in patients with cellular rejection shortly before or at the time of diagnosis of rejection as compared to the transplant control group. Levels of sCD8 were not significantly increased in patients with cellular rejection. We found a more pronounced elevation in the mean marker ratios of both sIL2R and sCD8 in patients with cytomegalovirus hepatitis which were higher compared not only to the transplant control group but also compared to the cellular rejection group. The rise of serum levels preceded the histologic diagnosis of cytomegalovirus hepatitis and detection of cytomegalovirus in blood cultures. Increased serum levels of sIL2R with concomitant elevation of sCD8 suggest the diagnosis of cytomegalovirus hepatitis over cellular rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗
PubMed1994

Phase I trial of combined recombinant interleukin-2 with levamisole in patients with advanced malignant disease.

A Phase I study of rIL-2 and levamisole was performed to evaluate the activity, toxicity, and effect on immune parameters of this combination of agents in patients with advanced malignancy. Twelve patients with advanced cancer were included and begun on therapy with rIL-2, 1 x 10(6) U/m2 subcutaneously (SQ) daily for 5 days and levamisole beginning at 25 mg/m2 orally three times daily for 5 days. The dose of levamisole was increased to 50 mg/m2 thrice daily during this study. Immune parameter analysis included the percentages of lymphocyte subsets in peripheral blood, cellular cytotoxicity assays versus K562 and Daudi cells, and lymphocyte blastogenesis to the recall antigens tetanus toxoid and Candida albicans. The dose-limiting toxicities were pruritus, nausea, and facial edema. There were no indications of significant hematologic or hepatorenal toxicities. No patient fulfilled the traditional criteria for an objective response. In 8 of 9 patients with immune parameter data available there was an increase in cellular cytotoxicity and in the percentage of lymphocytes with the natural killer phenotype (CD3-, CD16/56+). This regimen can be given as an outpatient with acceptable toxicity. For Phase II investigations the doses of rIL-2, 1 x 10(6) U/m2 SQ daily x 5 days and levamisole, 50 mg/m2 three times daily x 5 days is recommended.

Adolescent↗
PubMed1994

Multi-centre comparison of ABBOTT MATRIX Aero to Pharmacia Standard RAST, Modified RAST and skin puncture tests.

ABBOTT MATRIX Aero is an enzyme immunoassay which measures specific IgE antibodies to 14 individually calibrated airborne allergens using a single serum specimen. In this study, ABBOTT MATRIX performance was evaluated in comparison to the results of skin puncture test and the Standard and Modified RAST procedures. The ABBOTT MATRIX demonstrated overall sensitivity of 89% vs. Standard RAST and Modified RAST, with specificity greater than 92% vs. both methods. ABBOTT MATRIX sensitivity vs. skin test (71%) exceeded that of the Standard and Modified RAST procedures (62% and 67% respectively). Positive results reported by ABBOTT MATRIX but not RAST were corroborated by skin test results for 3 of 5 allergens evaluated. All in vitro systems demonstrated specificity of approximately 90% vs. skin test. The ABBOTT MATRIX system provided results which compared favorably with the results of skin test and RAST, but required less hands-on time to obtain quantitative specific IgE measurements to multiple allergens.

Adolescent↗