The quantitative validation of non-endoscopic disease activity indices in ulcerative colitis.

BACKGROUND: Ulcerative colitis disease activity indices have not been formally validated. AIM: To analise quantitatively the psychometric and performance validity of two non-endoscopic indices for ulcerative colitis, the Simple Clinical Colitis Activity Index and the Seo Index. METHODS: In 66 patients with ulcerative colitis, the measurement of disease activity was repeated with the two non-endoscopic indices, St Mark's Index, and the Inflammatory Bowel Disease Questionnaire. Psychometric validity was evaluated by measuring the content, construct, criterion-convergent and criterion-predictive validity on a 0-1 scale. Performance validity was evaluated by measuring the reproducibility and responsiveness on a 0-1 scale. RESULTS: The Simple Clinical Colitis Activity Index had good to excellent psychometric and performance validity, while the Seo Index had moderate to excellent psychometric validity and moderate to good performance validity. The Simple Clinical Colitis Activity Index had weaknesses in content validity and in responsiveness. The Seo Index had weaknesses in content validity, construct validity and responsiveness. CONCLUSIONS: These two non-endoscopic indices for ulcerative colitis have good psychometric and performance validity, and are now the most rigorously validated disease activity indices for ulcerative colitis. The Simple Clinical Colitis Activity Index appears to have better overall validity. Quantitative evaluation identifies weaknesses in disease activity indices, and can lead to better disease activity indices for ulcerative colitis.

Patient defined dichotomous end points for remission and clinical improvement in ulcerative colitis.

BACKGROUND AND AIMS: Ulcerative colitis disease activity indices offer good statistical power but small changes in these indices may not be clinically important. There are no validated definitions of remission or of significant improvement for these indices. The use of clinically important end points would strengthen the validity of study outcomes. Our aims were to identify objective end points in standard disease activity indices for remission and for improvement in ulcerative colitis. METHODS: Sixty six consecutive patients with ulcerative colitis provided information about remission status and their disease activity. At a return visit 1-14 months later, these patients provided information about the change in their disease activity, and non-invasive indices were measured. RESULTS: Specific objective end points for determining remission with four standard indices and a quality of life instrument were determined (St Mark's <3.5, ulcerative colitis disease activity index <2.5, simple clinical colitis activity index (SCCAI) <2.5, Seo <120, and inflammatory bowel disease quality of life index (IBDQ) >205). These cut offs also identified patients who met a regulatory definition of remission. Specific objective end points for clinical improvement in two non-invasive indices and a quality of life instrument were determined with good sensitivity and specificity (SCCAI decrease >1.5, Seo decrease >30, IBDQ increase >20). CONCLUSIONS: We found specific cut off values for disease activity indices that identify patients who have significantly improved or achieved remission in an objective, sensitive, and specific manner. These cut offs should help in the interpretation of the outcomes of clinical trials in ulcerative colitis.

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease.

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.

IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease.

This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen alpha1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, alpha-smooth muscle actin (A), vimentin (V), desmin (D), and c-kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and "normal" ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen alpha1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V(+) cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V(+)/A(-)/D(-)), myofibroblasts (V(+)/A(+)/D(+)), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V(-)/A(+)/D(+)). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver.

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.

Intestinal metabolism and transport of 5-aminosalicylate.

The purpose of this study was to determine the characteristics of intestinal absorption and metabolism of 5-aminosalicylic acid (5ASA). Regional perfusions of 5ASA in the anesthetized rat resulted in the appearance of N-acetyl-5-aminosalicylic acid in the intestinal lumen. Lumenal metabolite appearance was proportional to 5ASA permeability, which was 5-fold higher in the jejunum than in the ileum. Intestinal elimination significantly decreases 5ASA absorption at low lumenal drug concentrations and this process is saturated at high drug concentrations. Metabolite levels in intestinal tissue were higher than plasma levels at low perfusion drug concentrations, whereas the reverse was observed at high concentrations. Transport and metabolism of 5ASA was studied in Caco-2 monolayers. At low drug concentrations, 5ASA was preferentially transported in the basolateral (BL) to apical (AP) direction. With 5ASA incubation in either the AP or BL chamber, the N-acetyl metabolite appeared only in the AP compartment. Transport of N-acetyl-5-aminosalicylic acid was also exclusively observed in the BL to AP direction. Clinical data indicate that anti-inflammatory response to oral 5ASA correlates with the amount of 5ASA delivered to the intestinal tissue. This study shows that at lumenal levels below 200 microg/ml (concentrations that are typically achieved by controlled release dosage forms), intestinal secretion of 5ASA accounts for more than 50% of the total elimination and can significantly affect tissue levels and, therefore, may be an important factor in determining the response to 5ASA therapy.

Nonviral vector-mediated thymidine kinase gene transfer and ganciclovir treatment in leiomyoma cells.

OBJECTIVE: To test the hypotheses that ganciclovir is cytotoxic to leiomyoma cells transfected with herpes simplex virus thymidine kinase and that estrogen modulates the responsiveness of tumor cells to this gene therapy approach. METHODS: Human and rat cultured uterine leiomyoma cells were transfected with plasmids encoding the beta-galactosidase gene, thymidine kinase gene, or a control plasmid. Transfection efficiency was monitored by measuring beta-galactosidase enzyme activity. Ganciclovir cytotoxicity in thymidine kinase-transfected cells was assessed by monitoring cell viability using trypan blue exclusion. The "bystander effect," a phenomenon in which thymidine kinase-expressing cells exposed to ganciclovir are toxic to adjacent thymidine kinase-nonexpressing cells, was assessed when thymidine kinase vector-transfected cells were cocultured with control plasmid-transfected cells at various percentages before exposure to ganciclovir. The effect of estradiol on ganciclovir-thymidine kinase-mediated cytotoxicity was assessed in estrogen-responsive rat leiomyoma cells. RESULTS: A thymidine kinase-ganciclovir-mediated "bystander effect" was demonstrated, with 48.6% (human) and 65.6% (rat) cell death when 5% of the leiomyoma cells were transfected with the pNGVL1-tk vector, with 0.84% and 1.9% of the cells expected to express thymidine kinase as based on the 16.7% and 39.8% transfection efficiency determined by the reporter gene assay in human and rat leiomyoma cells, respectively. Estradiol promoted cell growth and enhanced the "bystander effect" in rat leiomyoma cells. CONCLUSION: These findings demonstrate the feasibility of using thymidine kinase gene therapy as a novel treatment for uterine leiomyomas. The effect of estrogen may provide a mechanism to enhance the tumor-suppressive effect of this approach.

IGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth muscle.

Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in intestines of rats with peptidoglycan-polysaccharide enterocolitis by Northern analysis and in situ hybridization. IGFBP-5 mRNA was increased 2.4 +/- 0.5-fold in inflamed rat colon compared with controls and was highly expressed in smooth muscle. Cultured rat intestinal smooth muscle cells were used to study the regulation of IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased IGFBP-5 mRNA (1.9 +/- 0.1-fold) and collagen type alpha1(I) mRNA (1.6 +/- 0.2-fold) in cultured smooth muscle cells. IGF-I induced a dose- and time-dependent increase in IGFBP-5 in conditioned medium by Western ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5 mRNA decay rate after transcriptional blockade. Cycloheximide abolished IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by intestinal smooth muscle and is increased during chronic inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in intestinal smooth muscle cells.

Substance P as a mediator of colonic secretory reflexes.

The role of substance P in neural reflex pathways activated by stroking was investigated in muscle-stripped segments of distal colon from guinea pigs. Stroking the mucosal surface with a brush at 1 stroke/s evoked an increase in short-circuit current (Isc) indicative of chloride secretion. The response to mucosal stroking was maximally reduced by 69-75% by the antagonist GR-82334. The agonist [Sar9,Met(O2)11] substance P caused a bumetanide-sensitive increase in Isc when added to the mucosal or serosal bath. Ablation of extrinsic afferents with acute or chronic administration of capsaicin did not alter the mucosal stroking response. Reverse transcription-polymerase chain reaction and in situ hybridization revealed the presence of neurokinin1 (NK1) receptor messenger RNA in isolated colonocytes or crypt glands. Ligand binding of 125I-Bolton-Hunter-labeled substance P was inhibited by GR-82334. The 50% inhibitory concentration was 0.84 nM. The results demonstrate a role for substance P released from capsaicin-insensitive submucosal neurons and in mucosal stroking reflexes. The presence of NK1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells.

Insulin-like growth factors and inflammatory bowel disease.

Hallmarks of IGF-I action include synergy with other hormones and growth factors and the ability to stimulate proliferation or differentiated cell function dependent on physiological or pathophysiologial context. A complete understanding of IGF action in IBD will require analyses of mechanisms of IGF interaction with other growth factors, hormones and cytokines. GH and IGF-I may be administered to children over prolonged periods to correct growth disorders. The definition of the benefits and problems of GH/IGF-I therapy in IBD needs to distinguish between long-term and short-term effects. Short-term administration of GH and IGF-I to animal models of IBD such as the PG-PS and TNBS models, which share features of Crohn's disease (Sartor, 1992), and a recently developed murine model of ulcerative colitis induced by ingestion of dextran sulphate (Okayasu et al, 1990; Sartor, 1992; Cooper et al, 1993) could address the beneficial or detrimental consequences of short-term GH/IGF-I therapy. Adaptation of the PG-PS, TNBS and dextran sulphate models of inflammation to available transgenic mouse lines that over-express GH and IGF-I (Behringer et al, 1990; Ulshen et al, 1993), especially if over-expression is inducible, could help to define the potential benefits and problems of long-term GH/IGF-I therapy or the effects of GH/IGF-I on immune cell function and cytokine production during intestinal inflammation. It will be useful to study intestinal inflammation and complication in animal models of GH or IGF-I deficiency. In this regard, mice with targeted ablation of the IGF-I gene could be useful (Liu et al, 1993) although neonatal mortality in these models currently poses problems for in vivo studies. Development of mesenchymal cell lines from such animals could, however, provide a useful in vitro system to study the role of IGF-I in altered cell function in response to pro-inflammatory cytokines.

Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats.

BACKGROUND/AIMS: Subserosal injection of purified group A streptococcal peptidoglycan-polysaccharide (PG-APS) induces chronic relapsing granulomatous enterocolitis and systemic inflammation in susceptible inbred Lewis rats but only transient intestinal injury in Buffalo and Fischer rats. Cecal interleukin 1 (IL-1) and IL-1 receptor antagonist (IL-1ra) expression was measured in inbred rats displaying differential susceptibility to experimental enterocolitis. METHODS: The ileum and cecum of Lewis, Buffalo, and Fischer rats were subserosally injected with purified PG-APS or albumin. IL-1 and IL-1ra messenger RNA (mRNA) and protein (IL-1 only) were measured 1 or 27 days later. PG-APS-injected Lewis rats were treated with recombinant human IL-1ra. Kinetics of IL-1 and IL-1ra mRNA expression were studied in peritoneal cells. RESULTS: All rats strains developed acute inflammation with increased cecal concentrations of IL-1 beta and IL-1ra mRNA. Lewis rats developed chronic enterocolitis and had higher IL-1 and IL-1ra mRNA tissue levels than Buffalo or Fischer rats, which displayed no chronic inflammation. IL-1 beta and IL-1ra were produced by submucosal granulomas and correlated with inflammation. IL-1 alpha protein levels paralleled IL-1 beta mRNA expression. IL-1ra treatment attenuated acute and chronic enterocolitis, adhesions, and arthritis. PG-APS induced IL-1 and IL-1ra expression in peritoneal cells from Lewis and Fischer rats. CONCLUSIONS: Bacterial cell wall polymers stimulate IL-1 and IL-1ra expression in vivo and in vitro. These counterbalancing cytokines are increased in experimental enterocolitis and have important immunoregulatory roles in intestinal inflammation.

Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain.

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and 35S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain.

Insulinlike growth factor I and interleukin 1 beta messenger RNA in a rat model of granulomatous enterocolitis and hepatitis.

BACKGROUND: Insulinlike growth factor I (IGF-I) is mitogenic for fibroblasts and smooth muscle cells and stimulates collagen synthesis. The present study tested the hypothesis that IGF-I is important in the development of granulomatous inflammation and fibrosis. METHODS: IGF-I messenger RNA (mRNA) was measured in bowel and liver of rats with peptidoglycan-polysaccharide-induced chronic granulomatous enterocolitis and hepatitis using RNase protection. Cellular sites of IGF-I mRNA and IGF-I peptide precursor were localized by in situ hybridization and immunohistochemistry, respectively. Sites of IGF-I synthesis were compared with sites of interleukin 1 beta mRNA expression. RESULTS: IGF-I mRNA was increased 3.7-fold in cecal tissue from peptidoglycan-polysaccharide-injected rats compared with controls. IGF-I mRNA was up-regulated in fibroblastlike cells in the intensely fibrotic periphery of cecal and hepatic granulomas. This region also expressed IGF-I peptide precursor. Interleukin 1 mRNA localized to macrophage-like cells in the center of granulomas. CONCLUSIONS: IGF-I may be important in the development of fibrosis in this model of Crohn's disease. The localization of IGF-I and interleukin 1 mRNAs to distinct but adjacent sites is consistent with a paracrine interaction between cells expressing IGF-I and interleukin 1.

Enhanced growth of small bowel in transgenic mice overexpressing bovine growth hormone.

BACKGROUND: Transgenic mice with a bovine growth hormone gene linked to a mouse metallothionein I promoter (growth hormone transgenics) are a model of chronic growth hormone excess. METHODS: Growth of small bowel mucosa in ad libitum-fed growth hormone transgenics and wild type littermates and in growth hormone transgenics pair fed with wild-type littermates were compared. RESULTS: In both groups, body weight and small bowel weight were greater in growth hormone transgenics. Similarly, mucosal mass was 50%-100% greater in growth hormone transgenics, and the effect was greatest in proximal bowel. Villus height, measured in jejunum, was also greater in growth hormone transgenics. Measurements of mucosal proliferation did not differ between the growth hormone transgenics and wild type. Abundance of insulin-like growth factor-I messenger RNA in bowel was greater in growth hormone transgenics. CONCLUSIONS: Chronic growth hormone excess results in increased growth of small bowel mucosa. This effect appears to be specific because it occurred in ad libitum-fed and diet-restricted growth hormone transgenics, influenced villus height, and was more pronounced in upper than lower small bowel. The effect of chronic growth hormone excess does not appear to be secondary to an increase in the rate of mucosal proliferation, suggesting an effect on lifespan of mucosal cells.

Immunotherapy with short ragweed fraction A:D-glutamic acid:D-lysine polymer in ragweed hay fever.

We report the first human trial of immunotherapy employing the nonimmunogenic carrier, D-glutamic acid:D-lysine linked to short ragweed (SRW) fraction A (fraction A:D-GL). Twelve SRW-sensitive patients with no immunotherapy during the previous 19 yr received a 2-mo (7/79 to 9/79) course of fraction A:D-GL (average dose 49.5 mg, range 21 to 78 mg). We compared their symptom scores and serologic changes with two control groups of SRW-sensitive patients. Patients receiving fraction A:D-GL demonstrated at least a tenfold decrease in skin-test sensitivity to SRW and had statistically lower mean seasonal symptom scores (p less than 0.02) than untreated controls. Mean seasonal symptom scored did not differ statistically from those of control patients on year 4 of immunotherapy. In contrast to the expected suppression of IgE, we found that fraction A:D-GL stimulated both IgE and IgG responses to SRW and SRW-antigen E. These increases in IgE and IgG antibodies were significantly greater than in the control groups and appeared to be due largely to injection of fraction A:D-GL. Though fraction A:D-GL was generally well tolerated, we noted mild generalized urticaria in three patients, and large local reactions in five others. The difference between our results and the earlier results in mice may reside in the particular characteristics of this preparation of fraction A:D-GL.

Preseasonal intranasal immunotherapy with nebulized short ragweed extract.

We determined the effect of preseasonal intranasal short ragweed (SRW) immunotherapy in a double-blind, nonpaired, 20-wk study involving 33 SRW-sensitive patients. Patients were selected on the basis of an elevated IGE serum antibody level, a positive intradermal skin test, and a positive intranasal challenge to SRW antigen. SRW-treated patients sprayed SRW solutions intranasally six times a day for 12 wk preseasonally. Placebo-treated patients used nebulized solutions containing buffer or histamine that were interchanged randomly throughout this period. The SRW-treated group reported more preseasonal symptoms than the placebo-treated group (p less than 0.003); however, during the SRW pollination season, the SRW-treated group reported significantly less sneezing, nasal congestion, rhinorrhea, red/itchy eyes, itchy nose/throat, and cough/wheeze. Supplemental antihistamine usage was similar in both groups. The treatment did not affect serum IgE antibody levels to crude SRW, AgE Ra3, or Ra5 in either group at any time during the study. No significant production of IgG antibody to SRW was seen in either group. One SRW-treated patient developed acute sinusitis after 2 wk of treatment; otherwise no side effects other than symptoms of hay fever were noted. Although intranasal SRW immunotherapy may offer an effective and less costly alternative to parenteral immunotherapy, reduction in hay fever symptoms during the pollination season was achieved at the expense of provoking these symptoms during the preceding weeks.

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test.

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.

Interference in ragweed pollen and honeybee venom radioallergosorbent tests.

We studied sera from patients sensitive to short ragweed (SRW) and honeybee venom (HBV) to investigate serum factors able to interfere with the measurement of IgE antibody levels by the radioallergosorbent test (RAST). We heated sera to destroy IgE antibodies and tested them for interference in the RAST. Heating sera for 4 hr at 56 degrees C destroyed up to 98% of the IgE antibody activity. After immunotherapy sera from patients sensitive to SRW and HBV produced striking interference in the RAST. The interference was most marked in the RAST employing 50-microgram quantities of microcrystalline cellulose-linked allergens, but it was also evident in the RAST employing 500-microgram quantities of such allergens and in the commercial RAST in which allergen is linked to paper disks. The interfering substance eluted from diethylaminoethyl (DEAE) cellulose in the IgG fraction. The interference could be eliminated by increasing the relative quantity of solid-phase allergen; RAST interference was not detected when SRW extract was linked to Sepharose 4B. The results indicate that serum factors, presumably IgG antibodies, produce interference in the RAST. Thus immunotherapy studies that measure IgE antibody levels by the RAST must consider the possibility that IgG antibodies can appear to depress IgE antibody levels. Furthermore, because commercially available RAST disks are susceptible to RAST interference, they must be used with caution for the diagnosis of allergy in patients whose sera may contain significant quantities of IgG antibodies.